Controlling Reactivity and Electron Transfer in De Novo Designed Artificial Cu Proteins by Systematic Primary, Secondary, and Outer Sphere Modulation

doi:10.21203/rs.3.rs-4714368/v1

Download PDF

Article

Controlling Reactivity and Electron Transfer in De Novo Designed Artificial Cu Proteins by Systematic Primary, Secondary, and Outer Sphere Modulation

https://doi.org/10.21203/rs.3.rs-4714368/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Copper-dependent metalloenzymes play essential roles in biology. However, unraveling how the active sites and the surrounding environment influence their functions presents a significant challenge. Inspired by Cu enzymes, we report de novo designed artificial copper proteins (ArCuPs) within trimeric (3SCC) and tetrameric (4SCC) self-assemblies, featuring a trigonal Cu(His)₃ and a square pyramidal Cu(His)₄(OH₂) coordination. 3SCC electrocatalyzes C-H oxidation, but 4SCC does not. Cu^I-3SCC reacts more rapidly with H₂O₂ compared to O₂, while 4SCC is less active. These trends mirror the peroxygenation of lytic polysaccharide monooxygenases (LPMOs) and the unreactive nature of the particulate methane monooxygenase (pMMO) Cu_B site. The differences in reactivity are attributed to inherent reducibility and reoxidation processes, with ET and reorganization energies (l) along with second-sphere and outer-sphere H₂O-mediated H-bonding patterns providing further insights. Modulation of second/outer-sphere H-bonding without changing the primary coordination tunes the solvent l, which renders the unreactive 4SCC active for C-H peroxidation.

Physical sciences/Chemistry/Inorganic chemistry/Bioinorganic chemistry/Metalloproteins

Biological sciences/Biochemistry/Peptides

De novo protein design

copper enzymes

bioinorganic chemistry

electron transfer

H-bonding

Redox-active metalloenzymes possess optimized primary, secondary, and outer-sphere interactions to control catalysis and electron transfer (ET).^1–5 The active site minimizes oxidation state-dependent structural changes, which lowers the reorganization energy (λ), facilitating ET and catalysis. The coordination number, geometry, and protein matrix are critical determinants facilitating redox catalysis. Copper (Cu)- -containing metalloproteins are at the heart of numerous biochemical processes, such as respiration, ET, biomass degradation, hydrocarbon oxidation, and the biosynthesis of hormones and cofactors.^{1, 6, 7} The type-I Cu centers are involved only in ET, but the type-2 Cu proteins can either participate in ET or catalysis.^{1, 6} Cu is predominantly found in a 3–5 coordinate geometry combining His, terminal amine, and oxygen-derived ligands such as Asp, Tyr, or water. Amine oxidase,⁸ nitrite reductase,⁹ and the Cu_B site of Cyt c oxidase¹⁰ feature a Cu(His)₃ ligation (Fig. 1A-B), also found in the coupled binuclear active site of tyrosinase (Ty). The lytic polysaccharide monooxygenases (LPMOs) have a T-shaped Cu(His)₂NH₂ geometry formed by a His-brace (Fig. 1C).^11–15 In addition to O₂, LPMOs can also utilize H₂O₂ as a terminal oxidant to break down recalcitrant cellulosic substrates, which makes them designated as peroxygenases.^16–21

Particulate methane monooxygenases (pMMOs) are membrane-bound enzymes that were initially thought to feature a bimetallic Cu-Cu active site.^{22, 23} However, based on recent structural and EPR studies, pMMOs are now thought to contain only mono-copper active sites.^24–27 The membrane-bound Cu_C or the Cu_D site is the proposed active site where O₂ binding and CH₄ oxidation occur.^25–27 Another Cu site, Cu_B, located in the soluble PmoB side, has a unique square pyramidal Cu(His)₃(NH₂)(OH₂) coordination from 3 His, a terminal amine, and axial water (Fig. 1D). Although not the hydrocarbon binding site, the Cu_B site is important since deleting Cu_B ligands in homologous hydrocarbon monooxygenase reduces the enzymatic activity by > 80%.²⁸ Besides being unreactive to exogenous substrates, the Cu_B site is also difficult to reduce.²⁵ Thus, the Cu_B appears to be a structural site but is still important for function.²⁵ The unreactive nature of the Cu_B is intriguing, given that many synthetic Cu complexes with tripodal tetradentate ligands can activate O₂ and H₂O₂.^29–32

How the structural differences translate to function that makes a CuN₃ site (LPMO, etc.) active to exogenous substrates but not the CuN₄ site (Cu_B of pMMO) remains poorly understood. Given that the active sites are embedded in diverse protein matrices of varying secondary and tertiary structures, it becomes challenging to attain a unified and generalized view of how the coordination number, ligand environment, and remote interactions influence ET, λ, and H₂O₂/O₂ reactivity, ultimately determining functional outcomes.

Governed by the primary sequence repeat pattern, the de novo protein design approach enables the creation of active sites within similar structural motifs, such as the α-helix, offering insights into how the immediate coordination environment of metalloenzymes determines their functions.^{3, 33–35} The knobs-into-holes (KIH) packing of hydrophobes provides a distinctive handle to produce self-assemblies^36–38 with controlled oligomeric states where an active site of interest can be introduced. Given the modular nature of the design, different types of coordination numbers and geometries can be constructed within de novo proteins, as dictated by self-assembly. Indeed, recent success has been achieved in creating functional artificial metalloenzymes (ArMs) within de-novo scaffolds.^39–48

One such example is the novo-designed artificial Cu proteins (ArCuPs) that function as peroxidases and peroxygenases featuring Cu(His)₃ active sites within trimeric (3SCC) self-assemblies.^{45, 46} The 3SCC ArCuP activates H₂O₂⁴⁵ and electrocatalyzes the 2e^- and 4e^- oxidation and peroxygenation.⁴⁴ To elucidate what factors control the physical and reactivity outcomes of CuN₄ vs. CuN₄ sites in biology that make the former reactive but the latter unreactive, we have designed a Cu(His)₄ site within a de novo designed tetrameric ArCuP (4SCC), akin to the Cu_B site of pMMO. We demonstrate how the differences in the coordination properties of 4SCC and 3SCC alter their O₂ and H₂O₂ activation as well as ET properties, which govern their peroxygenase function. The reduction and reoxidation kinetics probed by electronic and EPR spectroscopy suggest that 3SCC is easier to reduce and reoxidize than the 4SCC. The 4SCC is also unreactive to the peroxygenation of C-H substrates. To address how coordination differences influence ET properties and the resultant catalytic outcomes, the λ values of these ArCuPs have been experimentally determined. 4SCC has an inherently higher λ than 3SCC, which is significantly dominated by the solvent organization energy. Intriguingly, modulation of an H-bond involving the primary coordination-sphere His and a second-sphere Glu enables tuning of λ by altering the outer-sphere H₂O reorganization without affecting the primary coordination. The H-bond modulation and changes in the solvent reorganization also render the inactive 4SCC an active peroxidation catalyst.

2.1. Design of modular peptide assemblies: A hallmark of de novo protein design is the ability to construct self-assemblies with different oligomeric states by changing the primary sequence. This is achieved by the KIH packing of a and d site residues in the heptad (abcdefg)_n. For example, introducing an Ile at the a and d site of the heptad (IAAIKQE)_n produces the trimeric 3SCC ArCuP.⁴⁵ To design the tetrameric 4SCC assembly, the (LAAIKQE)_n motif was chosen, having a Leu at the a site and an Ile at the d site.⁴⁹ To introduce the His site in 4SCC, we chose the 9th position of the heptad, similar to the 3SCC, to allow a direct assessment of how the immediate coordination environment alters their physical and reactivity properties. The Ala residues favor the α-helix formation, while the Glu and Lys at e and g positions control the parallel orientation of the helices by ion pairs. An acetylated and amidated Gly residue caps the N- and C- termini, respectively. A Trp chromophore is introduced at an f site on the last heptad to aid in quantification. The final sequence of 4SCC is shown in Table 1.

Table 2

EPR parameters.
Sample		g_x, g_y	g_z	A_x,y (MHz)	A_z (MHz)	Ref
pMMO Cu_B		2.035 2.068	2.242	14, 30	570	ref²⁵
Cu^II-3SCC		2.05	2.25	116	566	ref⁴⁵
Cu^II-3SCC + H₂O₂		2.06	2.27	97	530	ref⁴⁵
Cu^II-A10W-3SCC		2.056	2.272	24	520	this work
Cu^I-3SCC + H₂O₂	80%	2.057	2.265	2	534	this work
Cu^I-3SCC + H₂O₂	20%	2.063	2.299	1	489	this work
Cu^I-3SCC + O₂	70%	2.059	2.276	8	525	this work
Cu^I-3SCC + O₂	30%	2.054	2.309	3	489	this work
Cu^II-4SCC		2.048	2.253	71 A_N = 51, 41, 44, 41	543 A_N = 55, 41, 44, 41	this work
Cu^IIA10W-4SCC		2.048	2.255	73 A_N = 49, 41, 44, 41	548 A_N = 53, 41, 45, 42	this work

2.2. Structure and electronic properties of 4SCC: 4SCC was crystallized in the Cu-bound form and diffracted to 1.36Å (statistics in Table S1). 4SCC crystallizes with a tetrameric quarternary structure produced by parallel assembly of the monomers (Fig. 2A), as designed. All four His residues point toward the helical axis and bind Cu exclusively via the N_ε atoms. A water molecule occupies an axial position at 2.6 Å, forming an approximate square pyramidal Cu(His)₄OH₂ coordination (Fig. 2B). The His residues are slightly distorted from the Cu plane with Cu-N_ε distances of 2.0–2.2 Å (Fig. 2C). In the Cu_B site of pMMO the Cu-N distances are in the range of 2.2–2.7 Å.²⁴ Two of the opposing N_ε atoms in 4SCC are located below Cu with an ∠N_ε-Cu-N_ε angle of 167° (Fig. 2D). The remaining two N_ε atoms lie above the Cu with an ∠N_ε-Cu-N_ε angle of 189°. The orientation of His C_ε atoms is also different, where the two of the opposing His residues are tilted away from each other at a greater extent (140° vs. 163°) than the others. The electronic spectrum of 4SCC has a d-d band (Fig. 2E) at ~ 600 nm. The EPR spectrum of 4SCC (Fig. 2F) is characteristic of a tetragonally elongated type-2 Cu center having an axial g tensor, with g_x,y = 2.048, g_z = 2.253 and resolved Cu hyperfine splittings of A_x,y = 71 MHz and A_z = 543 MHz (Table 2). Additional superhyperfine splittings from the ¹⁴N nuclei of four His ligands are observed with A_N(x,y) = 51, 41, 44, 41 MHz, and A_N(z) = 55, 41, 44, 41 MHz. The superhyperfine splittings suggest a slight asymmetry in Cu-ligand interaction, which is also consistent with differences in the Cu-His geometry in the X-ray structure. The observation of well-resolved Cu-¹⁴N(His) superhyperfine splittings suggests a high degree of structural homogeneity in the Cu coordination sphere of 4SCC. Finally, the axial symmetry (g_z > g_x = g_y) indicates that the unpaired electron resides in the d_x2−y2 orbital, pointing toward the four N ligands on the tetragonal plane.

2.3. Reactivity differences between 3SCC and 4SCC: 3SCC catalyzes H₂O₂ reduction electrochemically,⁴⁵ However, 4SCC does not, as no significant catalytic current corresponding to H₂O₂ reduction is observed with the latter (Fig. S1). 3SCC also electrocatalyzes the oxidation and peroxygenation of benzylic C-H substrates with catalytic proficiencies (1/k_TS = k_cat/K_M/k_uncat) in the range of 300–1200 M⁻¹. The catalytic proficiencies can be enhanced by outer-sphere steric modifications that affect catalysis by facilitating substrate access, influencing the nature of Cu-oxygen species, and improving stabilities.⁴⁶ On the contrary, 4SCC does not peroxygenate or oxidize these substrates. This is evidenced by the small catalytic current and charge (Fig. S2 A-B, blue) and a lack of product after electrolysis (Fig. S3). These results indicate that differences in metal coordination are significant determinants of their reactivity. This is consistent with the fact that Cu active sites with three protein-derived N ligands (e.g., LPMO, AO, Cu_B of CcO, NiR, etc.) are reactive, while the Cu_B site of pMMO, which has four protein-derived ligands, is not.

2.4. 3SCC is easier to reduce than 4SCC: Since the reactive Cu^I form is necessary for generating the Cu-oxygen intermediates,^{6, 16, 45, 46} we hypothesized that the inherent reducibility could influence the reactivity differences between 3SCC and 4SCC. To test this, we assessed the reduction kinetics using Trp fluorescence as a reporter of the Cu oxidation state. In the original designs, a Trp was present at the end of the peptide, which is too far from the Cu site to show any oxidation-state-dependent emission change. Thus, the Trp was moved to the 10^th position (A10W variants), one residue away from the Cu site (Fig. 3A-B, Table 1). These constructs also form the type-2 Cu sites (Fig. S4). Kinetics of Cu^IIà Cu^I reduction with ascorbate (Asc) shows that A10W-3SCC is reduced faster than A10W-4SCC (Fig. 3C, Fig. S5-S6). The rates vary linearly with [Asc] (Fig. 3D), yielding second-order rate constants of 1.9 ´ 10^-6 M^-1s^-1 and 7.6 ´ 10^-8 M^-1s^-1 for A10W-3SCC and A10W-4SCC, respectively

(Table 3). The two orders of magnitude rate difference suggests that the Cu(His)₃ motif is easily reduced than Cu(His)₄. This result supports our hypothesis that reactivity differences are influenced by the easy accessibility of the Cu^I form in a CuN₃ environment than in a CuN₄ environment, a conclusion consistent with the redox potentials (E°’) and experimentally determined λ of these ArCuPs (vide infra). In LPMO, the rate of reduction is fast and not rate-limiting.¹⁷ In

infra EPR). In contrast to Cu^II-3SCC, which produces the Cu^II-hydroperoxo species with H₂O₂,⁴⁵ the Cu^I reactivity is more complex (Scheme 1). In the peroxide pathway, the Cu^I--H₂O₂ association complex undergoes homolytic O-O bond cleavage to produce Cu^II-OH and ^·OH by Fenton-type chemistry. H-atom abstraction (HAA) by the ^·OH produces the oxyl Cu^II-O^· and H₂O. In the O₂-dependent pathway, cupric superoxo Cu^II-O₂^·- is produced initially, which, upon H⁺/e^- transfer, is converted to Cu^II-OOH.

The distal O protonation and ET produce the oxyl and H₂O, while the corresponding chemical event on the proximal O produces the Cu^I-H₂O₂ association complex, which enters the H₂O₂-dependent pathway. Thus, the reoxidation of Cu^I-LPMO with H₂O₂ can produce various Cu-oxygen species, such as Cu^II–OH---OH^·, Cu^II–O^·, or Cu^II–OH.¹⁷

The reaction of Cu^I-3SCC with H₂O₂ produces a prominent band at ~332 nm (Fig. 4A). A definitive assignment of this species is challenging due to many possibilities, as discussed above. In several model complexes, a Cu^II-hydroperoxo species appears in the range of 325-350 nm.^50-52 Although Cu^II-hydroperoxo species are typically prepared with Cu^II and H₂O₂ or from Cu^I+ O₂in the presence of an electron and proton source, Karlin and coworkers have reported a Cu^II-hydroperoxo species prepared from Cu^I and H₂O₂.⁵³ After the initial formation of Cu^II-OH and ^·OH from the homolytic cleavage, the ^·OH reacts with Cu^I to produce more Cu^II-OH, which then reacts with H₂O₂ to produce Cu^II-hydroperoxo and H₂O (Scheme S1). The Cu^I-H₂O₂ reaction also oxidizes the active site His,⁴⁵ which is observed with LPMO in the absence of substrate as well.^{16, 20} The reaction product of Cu^I-3SCC and H₂O₂ is best assigned as a Cu^II-oxygen species with a partially oxidized His.

The reaction of Cu^I-3SCC with O₂ is ~11-fold slower than with H₂O₂, producing a major band at ~336 nm plus a weaker feature at ~422 nm and another broad feature at ~540-570 nm (Fig. 4B). These features are typically observed for Cu-oxygen species, namely, Cu^II-hydroperoxo or Cu^II-alkylperoxo.^{54, 55} The ~575 nm feature is the Cu^II d-d band, suggesting the reoxidation of Cu^I^à^II with a peroxo ligation.

Cu^I-4SCC with both H₂O₂ and O₂ produces a similar species, having weak features at ~430 nm and 545 nm, plus a broad shoulder around 360 nm (Fig. 4C, D). In comparison to 3SCC, the 4SCC reacts with H₂O₂ and O₂ at ~40-fold and ~22-fold slower rates, respectively. This is consistent with the sluggish reactivity of 4SCC.

The reoxidation was also monitored by Trp quenching. An increased H₂O₂ (10 to 50-fold) concentration quenched the Trp signal (Fig. 4E-F) for A10W-3SCC, with a second-order rate (k_reox) of 1.6 ´ 10^-⁴M^-¹s^-¹ (Table 3). A higher H₂O₂ concentration is needed to reoxidize A10W-4SCC (Fig. 4F) with a k_reox of 2.8 ´ 10^-⁵M^-¹s^-¹. Analogous experiments with O₂ saturated solutions show that Cu^I-A10W-3SCC reoxidation is faster than Cu^I-A10W-4SCC (Fig. S7), with a pseudo-first-order rate of 0.0019 s^-1 for the former. No kinetic information was extracted for A10W-4SCC due to its poor reactivity with O₂.

Time-dependent EPR is further used to examine the reoxidation of Cu^I-ArCuPs with saturated O₂ solutions or with 100-fold excess H₂O₂ anaerobically. With O₂, a gradual reoxidation is observed during the monitored 1h timeframe (Fig. 5 A, C), producing a major species (~70%) with g_x,y of

2.059, g_z of 2.276, and A_z of 525 MHz (Table 2). The minor species have g values of 2.054, 2.309, and A_z of 489 MHz. With H₂O₂, the signal saturates within ~30 min (Fig. 5 B, C), further validating the faster reoxidation process than O₂. The major species (80%) produced with H₂O₂ have g_x,y of 2.057 and g_z of 2.265, and A_z of 534 MHz (Table 2), while the remaining 20% of the species have g values of 2.063, 2.299, and A_z of 489 MHz. In 4SCC, the reoxidation is much slower (Figs. S8-S9), as anticipated.

To summarize, 3SCC undergoes faster reoxidation, with the rates being higher with H₂O₂ than O₂. The results with 3SCC are thus supportive of the initial higher reaction rate of LPMOs with H₂O₂ than O₂,¹⁶ while the low reactivity of 4SCC resonates with the inertness of the Cu_B site of pMMO.²⁵

2.6. Redox properties of the ArCuPs: Protein-film voltammetry was used to determine the redox properties of the ArCuPs adsorbed on pyrolytic graphite electrode (PGE). Repeated CV scans show that the ArCuPs remain stable on the electrode surface (Fig. S10).⁴⁵ The cathodic and anodic peak currents vary linearly with the scan rate, suggesting a surface-controlled redox process. Both the ArCuPs have E°’ in a narrow range but with a resolvable difference of ∼16 mV, with E°’ of 256 mV for 3SCC (Fig. 6A red ⁴⁵) and 240 mV for 4SCC (Fig. 6A blue) in deaerated pH 6.5 buffer. These E°’s are similar to the range of other His-coordinated type-2 Cu proteins irrespective of their role as catalytic or ET sites.^{1, 6} The CV for 4SCC is broader than 3SCC, which suggests a slower ET and that dispersion effects⁵⁶ contribute to the redox process of the former.

2.7. 4SCC has a higher λ than 3SCC with a significant solvent contribution that is negligible in 3SCC: To reconcile the reactivity differences of the ArCuPs we have investigated their ET properties. As the Marcus equation (i) describes, ET is dependent on λ.^{57, 58}$\:{k}_{ET}=\frac{4{{\Pi\:}}^{2}{H}_{AB}^{2}}{h\sqrt{4{\Pi\:}\lambda\:{k}_{B}T}}\text{e}\text{x}\text{p}\left[-{\left({\Delta\:}{G}^{0}+\lambda\:\right)}^{2}/4\lambda\:{k}_{B}T\right]\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left(\text{i}\right)$

The total λ (λ_T) for metalloproteins is contributed by the inner sphere (λ_Cu) and the outer sphere (λ_OS) reorganization, which comprises the surrounding protein matrix (λ_P) and the solvent (λ_H2O) reorganization.^{4, 59, 60} The λ_Cu reports any geometrical changes associated with the redox event at the Cu site. Traditionally, λ for Cu proteins such as azurin (Az) has been determined by photoexcitation followed by flash-quench of Ru/Os modified Az⁶⁰ or pulse radiolysis,⁶¹ where disulfide radical anions transfer electrons to the Cu center. Electrochemistry is an alternative method to derive λ from Arrhenius's analysis of temperature-dependent ET rates.^62–65 We have employed two electrochemical methods to determine λ of the ArCuPs: rotating disk electrochemistry (RDE) and chronoamperometry (CA). In RDE, the ArCuPs are dissolved in solution, and their electrochemical response is measured as they diffuse to the electrode surface. Since the protein is in solution, λ measured from RDE has both the inner sphere (λ_Cu) and outer sphere components (λ_P + λ_H2O) and thus reports λ_T. In contrast, the λ measured by CA involves direct immobilization of the ArCuPs on the electrode surface. Therefore, contributions from the solvent reorganization are expected to be minimal compared to situations where the protein is freely diffusing in solution.^{62, 63} Thus, the λ determined using CA has contributions from the Cu site and any changes to the polypeptide matrix during ET. We denote this sum as λ_Cu−P. Naturally, the difference in λ_T measured by RDE and λ_Cu−P measured from CA will yield the solvent contribution, λ_H2O (λ_H2O = λ_T – λ_Cu−P).

In the RDE method, temperature-dependent linear sweep voltammograms (LSVs) are collected at different electrode rotation rates (Fig. 6B, Fig. S11-14). These data are then subjected to Koutecký-Levich (K-L) analysis (eq ii, Fig. 6B inset).⁶⁶ The intercepts of these graphs in the mass-transport region yield the ET rate (k_ET), which are used to construct Arrhenius plots (Fig. 6C) ln(k_ETT^−1/2) vs. T⁻¹ (eq iii, Tables S2, S3, S7). The slopes of Arrhenius plots yield λ_T. The λ_T thus measured is 0.34 eV for 3SCC (Table 4). In contrast, 4SCC has a much higher λ_T of 0.89 eV (Fig. 6D).

For CA, temperature-dependent CVs of the ArCuPs immobilized on electrodes are recorded to extract E°’s (Fig. S15, Tables S4, S8). These E°’s are used to determine the overpotentials (η) at each temperature. The CA experiments were performed at various applied potentials (E_app = E°’ ± η; η = 50, 100, and 150 mV) (Fig. S16) against η chosen on both anodic and cathodic sides of E°’. The ln(i) vs. time(t) plots are obtained at different temperatures (Fig. 6E, Fig. S17-S18), the slopes of which yield the heterogeneous ET rates, k_app (eq (iv)). These were then utilized to construct the Arrhenius plots (Fig. S19-S20) and obtain λ_Cu−P.⁶⁷ From this analysis, the average λ_Cu−P of 0.36 eV is obtained for 4SCC and 0.27 eV for 3SCC (Fig. 6F, Table 4). Thus, λ_H2O for 3SCC is 0.07 eV and 0.53 eV for 4SCC. Combined RDE and CA analysis implies that not only does the 4SCC have a higher λ_T than 3SCC, but a significant solvent contribution, λ_H2O of 0.53 eV, is present in 4SCC but not in 3SCC.

2.8. λ comparison between ArCuPs and Cu proteins: In classical Cu complexes, the λ is quite high, ~ 1.88–2.4 eV.⁶⁸ However, the protein fold significantly lowers λ.⁶⁹ In WT Az, the λ is 0.82 eV,^{60, 70} and between 0.70–0.75 eV in plastocyanin (Table 4).⁷¹ In Az mutants, the range is 0.67–0.98 eV.^{72, 73} In Cu_A-Az, the λ is 0.4 eV,⁷⁴ approximately half of the WT Az. The T1 Cu site of blue AxCuNiR has a λ of 0.77 eV, while that of the green AcCuNiR has 0.57 eV.⁷⁵ The type-2 Cu site of blue AxCuNiR has a λ of 1.6 eV, much higher than the type-1 Cu site of the same protein. The type-2 site of C112D-Az has a relatively high λ of 2.1–2.3 eV, attributed to a loss of ligand and an expansion of the coordination sphere upon reduction, leading to a more flexible structure.^{76, 77} Interestingly, the double mutant C112D/M121L-Az produced a T0 Cu site, which lowered the λ to 0.9–1.1 eV, suggesting a constrained Cu site in the latter.⁷⁶ An exception is Cu-Aβ16, which, despite being unstructured, has a low λ of 0.3 eV,⁷⁸ which is similar to 3SCC. This has been ascribed to a preorganized ET (POET) process where the Cu coordination undergoes minimal structural changes upon ET. In select cases of designed synthetic Cu complexes, λ is reported to be ~ 0.95⁷⁹ – 1.1 eV,⁸⁰ with λ_Cu of 0.4 eV in one example,⁸⁰ similar to 4SCC.

2.9. Correlating structural differences to ET and catalysis: A less saturated Cu(His)₃ coordination is expected to lower λ_Cu−P than a more saturated 4SCC. However, to rationalize the presence of a significant solvent reorganization in 4SCC, one must look beyond the immediate coordination sphere. Indeed, differences in the 2° and outer coordination sphere interactions exist in the ArCuPs. Specifically, the His9 residues in 4SCC participate in 2°-sphere H-bonding with Glu8 from a neighboring helix via the available N_δ atoms (Fig. 7A). No such interaction is observed in 3SCC (Fig. 7B), even though both peptides have the -GluHis- motif in their heptads. Twelve H₂O molecules constitute an extended outer-sphere H-bonding network in 4SCC involving Glu8 (Fig. 7A). These structural differences between 3SCC and 4SCC alter the physical properties of the ArCuPs, which are translated to their function.

First, the 2°-sphere H-bonding will increase the electron density on the imidazole ring, making the Cu^II state more stable than the Cu^I state. Indeed, the E°’ of 4SCC is lower than 3SCC (vide supra). Second, as seen here, the extended H₂O-mediated H-bonding network will increase the outer sphere contribution to λ_T. Therefore, the combined 1°, 2°, and outer sphere H-bonding involving H₂O molecules add to the higher λ_T in 4SCC, with the major contribution coming from the solvent reorganization. This observation is coherent with hybrid quantum mechanics/molecular mechanics (QM/MM) simulation on Az, where the solvent reorganization around His117 is proposed to contribute ~ 80% of the total λ.⁸² The

coordination environment of 3SCC, along with the absence of 2°-sphere and outer-sphere water-mediated H-bond, contributes to a significantly lower λ_T, with nearly all the contribution coming from the Cu active site. In 3SCC, a single Glu8-H₂O H-bond is present (PDB 7L33), but since Glu8 does not make an H-bond with primary sphere ligand His9, the effect of outer sphere solvent reorganization is expected to be negligible, which is indeed seen here. The slower reaction kinetics of 4SCC with O₂ or H₂O₂ and its lack of reactivity for C-H oxidation can be attributed to the fact that the Cu^I form is relatively difficult to access in this construct owing to a large reorganization accompanying redox state change, compared to 3SCC.

One factor for the unreactivity of the pMMO Cu_B site, even to polar solutes such as nitrite and its non-reducibility, could be the inherently high reorganization energy of this site. The Cu_B site, being present in the soluble PmoB subunit, also brings the possibility of solvent contribution to the reorganization energy. Indeed, several 2°-sphere and outer-sphere H-bonds involving amino acid residues and solvent molecules are present around the Cu_B site (Fig. 7D).²⁷ Although λ determination for the pMMO Cu_B site will be nearly impossible, given the presence of multiple mononuclear Cu sites, our de novo 4SCC system offers a plausible reason for this site’s unreactivity. The poor reactivity of CuN₄ sites in a protein or polypeptide framework is intriguing, as several synthetic Cu complexes with tripodal tetradentate ligands can activate O₂ and H₂O₂.^29–32 We believe one important factor governing this reactivity difference is higher outer-sphere reorganization energy in the biomolecular systems than in the synthetic systems, which lack ligand and H₂O-mediated extended H-bonding networks.

2.10. Deleting H-bonding interactions modulates λ _H2O and enables C-H oxidation: An important question in metalloprotein design is “Is it possible to selectively alter reorganization energy by changing 2° and outer sphere H-bonding but keeping the 1° coordination sphere intact”? To address this, we designed a mutant to delete the Glu-His H-bond of 4SCC by replacing the Glu8 (g site heptad) with Lys.

Consequently, the Lys6 (e site heptad) was changed to Glu to maintain the charge neutrality and e-g ion pairs. From RDE and CA (Fig. S21-24), the λ_T and λ_Cu−P determined for K6E/E8K-4SCC are 0.69 eV and 0.36 eV, respectively (Fig. 6D, F; Table 4). This result shows that although the λ_Cu−P is identical to the parent 4SCC, the λ_H2O is significantly reduced to 0.33 eV compared to 0.53 eV in 4SCC.

To investigate if the experimental λ values correlate to structure, we solved the X-ray structure of the double mutant (Fig. 7C). The Cu site is nearly identical to 4SCC (Fig. S25), although some disorder in the N-terminal region of chain D is present in the mutant. As designed, the 2°-sphere His-Glu H-bond is eliminated. This, in turn, has removed the H₂O-mediated outer-sphere H-bonds involving the Glu residue as it is now 3-residue away from the His. The only H₂O-mediated H-bond is seen between the axial Cu-bound H₂O and a second H₂O nearby (Fig. 7C). Since the Cu site in 4SCC is located within the hydrophobic core, the deletion of 2°-sphere H-bonding does not alter the Cu(His)₄ site, leading to a similar λ_Cu−P for both 4SCC and the mutant. However, deletion of the 2°-sphere H-bond removes the outer-sphere H₂O-mediated H-bonding besides the axial H₂O—H₂O H-bond, leading to a 0.2 eV lower λ_H2O in the mutant than 4SCC. The axial H₂O-mediated H-bond also contributes to a higher λ_H2O in the mutant than in 3SCC, which lacks any H₂O-mediated H-bond. These findings demonstrate that by protein design, it is possible to modulate λ_H2O via selective modification of 2° and outer-sphere H-bonding without perturbing the 1° coordination sphere.

Next, we probed whether deleting the extended hydrogen-bonding interactions would make the double mutant active toward C-H oxidation. Unlike 4SCC, K6E/E8K-4SCC produces a significant catalytic current for benzyl alcohol peroxidation (Fig. S2A) with H₂O₂. As a result, an appreciable amount of charge is observed in the CPE experiment (Fig. S2 B-C), leading to the detection of benzaldehyde in the GC (Fig. S3) with a catalytic proficiency of 500 M^− 1. This value is still lower than that of 3SCC (1200 M^− 1), suggesting that differences in the primary coordination sphere remain a major contributor to activity. However, with the same CuN₄ primary coordination, K6E/E8K-4SCC is catalytically active, while 4SCC is not. We attribute this to differences in 2° and outer coordination-sphere H-bonding interactions. The lack of such interactions in K6E/E8K-4SCC makes the construct more flexible, which lowers its λ_H2O and enables it to act as an active peroxidation catalyst.

In summary, these results demonstrate that protein design not only allows for controlled tuning of outer-sphere solvent reorganization energies while maintaining the primary coordination but also renders an inactive metalloprotein functional by modulating H-bonding interactions.

2.11. Inner-sphere vs. outer-sphere ET pathways: The reoxidation of Cu^I to Cu^II can occur via either the inner-sphere or the outer-sphere ET mechanisms. To distinguish between these two processes, the relative rates for outer-sphere ET derived from the Marcus equation (i) are compared with the rates of reoxidation of Cu^I-ArCuPs. Combined with the E^o’s for ArCuPs and the E^o’ for 1e⁻ reduction of O₂ to superoxide (O₂/O₂^•−), the rates of outer-sphere ET (k_ET) derived from the Marcus theory are 1.13 × 10⁻¹ s⁻¹ and 1.58 × 10⁻² s⁻¹ (for 1 mM O₂) for 3SCC and 4SCC, respectively (see the SI for details). These rates for outer-sphere ET are faster than the rate of reoxidation with O₂ measured from UV (1.2 x 10^− 5 s^− 1), implying that the outer-sphere mechanism of ET prevails in 3SCC where a one-step reoxidation of Cu^I-3SCC to Cu^II-3SCC occurs with O₂. Due to slower kinetics, a similar mechanism is expected in 4SCC as well. In contrast to the ArCuPs, the reoxidation in LPMO is proposed to follow an energetically demanding inner-sphere mechanism where a Cu^II-superoxide species is produced, which is quickly released by water to regenerate the resting state of the enzyme.¹² From DFT calculations, it has been suggested that this thermodynamically unfavorable inner-sphere mechanism is driven by the binding of superoxide to Cu^II-LPMO, where an end-on Cu^II-superoxide triplet is formed with favorable free energy, making the overall process feasible.

In summary, an Ile/Ile to Leu/Ile switch of the hydrophobic layers enables modulating the oligomerization behavior to design the ArCuPs, and structural and spectroscopic evidence supports the design. The 3SCC is easier to reduce and reoxidize than the 4SCC. Additionally, the 3SCC activates peroxide and oxidizes/peroxygenates C-H substrates, whereas the 4SCC does not. These chemical properties of 4SCC resemble the nonreactivity of the pMMO Cu_B site. Cu^I-3SCC is more rapidly reoxidized with H₂O₂ than with O₂, similar to the peroxygenase reactivity of LPMOs in the absence of a substrate. The reaction profile of H₂O₂ and Cu^I-3SCC is complex, starting with homolytic O-O bond cleavage, leading to the Cu^II-oxygen species. Conversely, the O₂ reaction produces features consistent with a Cu-hydroperoxo species via an outer-sphere ET process.

The reactivity differences between 3SCC and 4SCC can be explained by both inner-sphere and outer-sphere reorganization energies coupled with structural differences. A less-coordinatively saturated 3SCC has a lower λ_Cu−P than 4SCC. However, 4SCC has a significantly higher solvent contribution due to an extended H₂O-mediated H-bond network around the 2°-sphere Glu8. This Glu8 is also H-bonded to the 1°-sphere His. These interactions increase the structural rigidity of 4SCC, leading to high reorganization energy and making it less reactive than 3SCC. Notably, removing the 2°-sphere His9-Glu8 H-bond eliminates the extended H₂O-mediated H-bonding network, reducing the outer-sphere solvent contribution while maintaining the primary coordination. Consequently, the inactive metalloprotein 4SCC has been converted to an active peroxidation catalyst. To our knowledge, this has not been achieved in protein design prior to this work. Finally, our finding of a high outer-sphere solvent reorganization may explain the lack of reactivity of the pMMO Cu_B site, which also has an extended H-bonding network around Cu. The outer-sphere effects rationalize why CuN₄ sites in proteins are less reactive, whereas the reactive synthetic analogs lack such extended solvent-mediated outer-sphere interactions.

4.1 Materials and methods: All chemicals and reagents were used as received. Buffers were prepared in Milli-Q (Millipore) water, treated with Chelex-100 overnight, pH adjusted as necessary, and filtered. Tris(hydroxymethyl) aminomethane (Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-(N-morpholino) ethanesulfonic acid (MES), sodium acetate, NaCl and HCl were purchased from Fisher Scientific. The mixed buffers contained 20 mM each of MES, sodium acetate, HEPES, and TRIS at the desired pH. CuCl₂ (Alfa Aesar), NaOH (VWR), and H₂O₂ (VWR) stock solutions were made in water, and ascorbic acid (VWR) stock was made in the buffer.

4.2. Peptide synthesis and purification: Peptides were synthesized, purified, and identified by mass spectrometry as described.⁴⁵

4.3 X-ray crystallography: Solutions for Cu^II-4SCC were prepared by adding 1 equiv of CuCl₂ to 45 mg/mL apo peptide in basic water. K6E/E8K-4SCC was dissolved at 35 mg/mL in 20 mM Tris pH 8.5, followed by metallation. Crystals were grown using the hanging drop method at ambient temperature. Two µL of Cu^II-4SCC solution was mixed with an equal volume of the well solution containing 0.1 M Tris pH 8.5 and 2 M ammonium sulfate. For Cu^II-K6E/E8K-4SCC, 2 µL of this solution was mixed with 2 µL of the well solution containing 0.1 M Tris pH 8.5, 0.2 M MgCl₂.6H₂O, and 28% PEG 4000. The well volume was 1 mL. Crystals appeared within 7–20 days. Crystals were harvested and soaked with 30% PEG 400 as a cryoprotectant before flash-freezing in liquid N₂.

X-ray diffraction data were collected at the Life Sciences Collaborative Access Team (LS-CAT) of the Argonne National Laboratory, the Stanford Synchrotron Radiation Lightsource, and the FMX beamline at NSLS-II. Diffraction images were indexed and integrated in XDS,⁸³ and scaled in Aimless.⁸⁴ Data quality assessment and twinning analysis were performed by phenix.xtriage.⁸⁵ The 4SCC structure was solved using Phaser⁸⁶ in CCP4i⁸⁷ by molecular replacement using 3R4A⁸⁸ as the search model, followed by automatic model building in Buccaneer.⁸⁹ Refinement was performed using phenix.refine⁹⁰ with iterative model building and density fitting analysis in Coot.⁹¹ The K6E/E8K-4SCC was solved similarly, using 4SCC as the search model, and refined in Refmac5.⁹² Data validation was performed at the wwPDB OneDep System. The structure factors and coordinate files have been deposited with PDB code 8VHS for 4SCC and 9BQR for K6E/E8K-4SCC. Data collection statistics are in Table S1. Electron density maps were drawn in CCP4mg.⁹³

4.4. UV-vis spectroscopy: The UV − vis spectra were recorded on an Agilent 8454 or Cary 5000 spectrophotometer. All samples were prepared in 10 mM MES buffer at pH 6.5. All solutions were degassed individually before mixing and starting the experiments. For the kinetics experiments, 0.125 mM Cu-peptide solutions and 12.5 mM H₂O₂ were mixed in a septa-capped anaerobic cuvette (Starna Cells) followed by monitoring the kinetics for 120 min with 1 min intervals in-between scans. Cu^I-peptides were prepared by adding 1 equivalent Asc inside a glove box (MBraun) and incubating for 15 min. UV-vis was checked to ensure complete reduction. For kinetics with O₂, 0.25 mM Cu-peptides were mixed with an equal volume of an O₂-saturated buffer, and the kinetics started immediately. An O₂-filled balloon was used to maintain an O₂-saturated environment. Initial rates were calculated from the first 3 minutes of the kinetic run.

4.5. Fluorescence spectroscopy: Kinetics of Cu^II reduction with Asc and Cu^I reoxidation experiments with H₂O₂ were performed anaerobically by monitoring the Trp fluorescence for the A10W-3SCC and A10W-4SCC peptides in septa-capped fluorescence cuvettes (Starna Cells) using a PTI fluorimeter in 10 mM MES pH 6.5. For reduction kinetics, 0.125 mM Cu^II-peptides were mixed with different equiv of Asc followed by continuous spectral monitoring. Reoxidation experiments were performed with 0.125 mM Cu^I-peptides (prepared by adding 1 equiv Asc to Cu^II-peptides) and an increasing concentration of H₂O₂. Each run was monitored for 120–180 min or until saturation was obtained, which depended on H₂O₂ concentration. In these experiments, the Trp excitation wavelength of 280 nm was used. An emission wavelength of 340 nm for A10W-3SCC and 345 nm for A10W-4SCC was selected based on the highest emission intensity. The final sample volume in each experiment was 800 µL.

4.6. Analysis of reduction and reoxidation kinetics: For reduction kinetics, the normalized fluorescence vs. time plots were fitted with a single-exponential function to extract the first-order rate constants at varying Asc concentrations. These rate constants were plotted against Asc concentration and fitted to a linear equation. The slopes of the linear fits represent the second-order rate constants with respect to Asc. A similar procedure was used for the reoxidation kinetics, except that the second-order rate constants were derived against varying H₂O₂ concentrations. This analysis was not performed for reoxidation kinetics with O₂ because only O₂-saturated solutions were used where the O₂ concentration was kept constant.

4.7. EPR spectroscopy: EPR data were collected at 77K on a Bruker X-band (9.4168 GHz) EMXPlus Continuous Wave spectrometer at the School of Chemical Sciences EPR lab at the University of Illinois. Samples for EPR contained 0.5 mM ArCuPs in 100 mM MES pH 6.5 plus 25% glycerol as a glassing agent. A power of 10.02 mW was used with attenuation of 13 dB, receiver gain of 30 dB, modulation amplitude of 10 G, and modulation frequency of 100 kHz. All instrument parameters were kept constant for each set of experiments. Cu^I-peptides were made as described in section 4.4. For reoxidation, a 100-fold H₂O₂ was added, and at different time intervals, samples were aliquoted to glycerol-containing EPR tubes and flash-frozen in liquid N₂ outside the glove box. For reoxidation experiments with O₂, 1 mM Cu^I-peptides were mixed with an equal volume of O₂-saturated buffer, and the reaction was allowed to proceed under an O₂ atmosphere outside the box with stirring. At different time intervals, samples were flash-frozen. Baseline correction of the raw data was done in Origin. The spin concentration of peptide samples was calculated from double spectral integration. Due to extensive superhyperfine features, reliable spin integrations were not obtained for 4SCC.

EPR simulations were done in EasySpin 6.0.0⁹⁴ using the MATLAB (R2024a) interface. An axial g tensor was used for Cu with the appropriate number of ¹⁴N nuclei (3 for trimer and 4 for tetramer). The hyperfine tensor Aframe was invoked to transfer the molecular frame to the A tensor. A Lorentzian line broadening was used. All simulations were done using the method suitable for frozen solutions (pepper).

4.8. Electrochemistry: All electrochemistry experiments were performed using a Wave Driver 20 bi-potentiostat (Pine Research) with pyrolytic graphite (PGE) as the working electrode, a coiled platinum wire as the counter electrode, and Ag/AgCl (in saturated KCl) as the reference electrode. The potential values were referenced against Ag/AgCl and converted to SHE. Temperature-dependent experiments were done in an RRDE cell with a water jacket by controlling the temperature from a circulating water bath.

The rotating disk electrode technique was used to determine the total reorganization energy λ_T of ArCuPs. All RDE experiments were performed with 0.25 mM Cu-peptides dissolved in 20 mM mixed buffer, pH 6.5, and 100 mM KCl as supporting electrolytes under anaerobic conditions. The cyclic voltammograms (CVs) were recorded in a solution for the Cu-peptides with PGE as the working, Ag/AgCl as the reference, and Pt as the working electrode. CVs at different scan rates show a linear dependence of the peak currents with root over scan rates, indicating that the process is diffusion-controlled. RDE experiments show an increase in the current with an increase in rotation rates following the Koutecký-Levich (K-L) relationship (eq ii), with i_L and i_K representing the Levich current and kinetic currents, respectively. Here, n is the number of electrons transferred during the redox process, F is Faraday’s constant 96485 C mol^− 1, A = area of the electrode (0.19625 cm²), C₀ is the concentration of Cu-peptide in solution (0.25 mM), and Γ is the surface coverage of the electrode (88 ± 8 x 10^− 12 mol cm^− 2). k_ET is the ET rate constant (k_ET in M^− 1s^− 1) from electrode to Cu-peptide in solution. D₀ is the diffusion coefficient of the analyte, ω is the angular velocity of the electrode, and ν is the kinematic viscosity of the solution.

$$\:\frac{1}{i}=\frac{1}{{i}_{L}}+\frac{1}{{i}_{K}}=\frac{1}{0.62nF{AC}_{0}{\left({D}_{0}\right)}^{2/3}{}^{-1/6}{}^{1/2}}+\frac{1}{nF{A{C}_{0}k}_{ET}}\:\:\:\left(ii\right)$$

Plots of inverse current vs. ω^−1/2 are linear. From the intercepts of these plots, the second-order rate constant for ET (k_ET) is determined given that in the absence of other competing chemical/redox phenomena, the only process occurring is the ET from the electrode to the ArCuPs. The k_ET was obtained at five different potentials chosen in the mass transport-limited region between −0.05V and 0.4V. The experiments were performed at three different temperatures, and for each temperature, k_ET was determined (Tables S2-S3). λ_T was obtained from the slopes of ln(kT ^− 1/2) vs. T^− 1 of the Arrhenius equation (iii).

$$\:\:\:\:\:\:\:\:\:\:\:\:\:\lambda\:=-4.03\:d{ln}\left({kT}^{-\frac{1}{2}}\right)/d\left[{T}^{-1}\right)\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left(\text{i}\text{i}\text{i}\right)$$

The CV and CA experiments for protein film electrochemistry were performed with 10 µL of 0.25 mM ArCuPs drop-cast on PGE followed by 6 µL of 0.1 M polymyxin B to facilitate electrostatic interaction between the ArCuPs and the electrode. The protein solution was then dried under N₂, and data were recorded in N₂-saturated 20 mM mixed buffers. Each CA experiment was performed for a total duration of 0.3 seconds. Temperature-dependent CV and CA experiments were performed at seven different temperatures between 280K and 310K with 5K increments. For temperature-dependent CA experiments, six different overpotentials (η) were chosen according to E^o’ from the temperature-dependent CVs. Three overpotentials were chosen on the cathodic (−150mV, −100mV, −50 mV) and three on the anodic (50 mV, 100 mV, 150 mV) side of E^o’s at each temperature. At a particular η, the current follows an exponential relationship with time according to eq (iv)

$\:i=\:{k}_{app}\:Q\:\text{e}\text{x}\text{p}(-{k}_{app}t)$ (iv)

where k_app is the apparent ET rate constant in s^− 1, Q is the total charge passed during CA, and t is the time. The decay current response is a combination of two processes: charging current decay and Faradaic current. The capacitive current decays completely within 1 ms for the blank electrode at pH 6.5. To avoid the charging current decaying completely from the Faradaic response, ln(i) vs. t plots were constructed beyond 1.2 ms (according to eq iv). From the slope of these semilogarithmic graphs, k_app (s^− 1) was determined at each temperature and η, which was used to determine λ (Tables S5-S6, S9). According to the Arrhenius equation (iii) obtained from the Marcus density of states, plots of ln(k_appT^−1/2) vs. T⁻¹ were made using k_app from above. The λ for the ArCuPs at each η was calculated from the slopes of these plots.

k _cat, K_M, and k_uncat for K6E/E8K-4SCC were determined as previously reported⁴⁶ for the 2e⁻ oxidation of benzyl alcohol to benzaldehyde. The calculated values for these parameters are 1.31 s^− 1, 37.3 mM, and 0.07 s^− 1, respectively.

AUTHOR INFORMATION

Present address: ^±Weill Cornell Medicine, 1300 York Avenue, NY 10065, USA

^xRegeneron Pharmaceuticals, Terrytown, NY 10591, USA

Corresponding Author: *[email protected]

Notes: The authors declare no competing financial interest.

ACKNOWLEDGEMENT

S.C. thanks the University of Mississippi for support. Major funding for the Bruker EMXPlus was provided by the National Science Foundation Award 1726244 (2017) to the School of Chemical Sciences EPR lab at the University of Illinois. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P30GM133894). This research used resources of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under contract no. DE-SC0012704. The Center for BioMolecular Structure (CBMS) is primarily supported by the National Institutes of Health, National Institute of General Medical Sciences (NIGMS) through a Center Core P30 Grant (P30GM133893), and by the DOE Office of Biological and Environmental Research (KP1605010).

Liu, J., Chakraborty, S., Hosseinzadeh, P., Yu, Y., Tian, S., Petrik, I., Bhagi, A., and Lu, Y. (2014) Metalloproteins containing cytochrome, iron–sulfur, or copper redox centers, Chem. Rev.114, 4366-4469.
Van Stappen, C., Deng, Y., Liu, Y., Heidari, H., Wang, J.-X., Zhou, Y., Ledray, A. P., and Lu, Y. (2022) Designing artificial metalloenzymes by tuning of the environment beyond the primary coordination sphere, Chem. Rev.122, 11974-12045.
Koebke, K. J., Pinter, T. B., Pitts, W. C., and Pecoraro, V. L. (2022) Catalysis and electron transfer in de novo designed metalloproteins, Chem. Rev.122, 12046-12109.
Winkler, J. R., and Gray, H. B. (2014) Electron Flow through Metalloproteins, Chem. Rev.114, 3369-3380.
Moser, C. C., Keske, J. M., Warncke, K., Farid, R. S., and Dutton, P. L. (1992) Nature of biological electron transfer, Nature355, 796-802.
Solomon, E. I., Heppner, D. E., Johnston, E. M., Ginsbach, J. W., Cirera, J., Qayyum, M., Kieber-Emmons, M. T., Kjaergaard, C. H., Hadt, R. G., and Tian, L. (2014) Copper Active Sites in Biology, Chem. Rev.114, 3659-3853.
Ciano, L., Davies, G. J., Tolman, W. B., and Walton, P. H. (2018) Bracing copper for the catalytic oxidation of C–H bonds, Nat. Catal.1, 571-577.
Adelson, C. N., Johnston, E. M., Hilmer, K. M., Watts, H., Dey, S. G., Brown, D. E., Broderick, J. B., Shepard, E. M., Dooley, D. M., and Solomon, E. I. (2019) Characterization of the Preprocessed Copper Site Equilibrium in Amine Oxidase and Assignment of the Reactive Copper Site in Topaquinone Biogenesis, J. Am. Chem. Soc.141, 8877-8890.
Suzuki, S., Kataoka, K., and Yamaguchi, K. (2000) Metal coordination and mechanism of multicopper nitrite reductase, Acc. Chem. Res.33, 728-735.
Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamaguchi, H., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., and Yoshikawa, S. (1995) Structures of Metal Sites of Oxidized Bovine Heart Cytochrome c Oxidase at 2.8 Å, Science269, 1069-1074.
Meier, K. K., Jones, S. M., Kaper, T., Hansson, H., Koetsier, M. J., Karkehabadi, S., Solomon, E. I., Sandgren, M., and Kelemen, B. (2018) Oxygen Activation by Cu LPMOs in Recalcitrant Carbohydrate Polysaccharide Conversion to Monomer Sugars, Chem. Rev.118, 2593-2635.
Kjaergaard, C. H., Qayyum, M. F., Wong, S. D., Xu, F., Hemsworth, G. R., Walton, D. J., Young, N. A., Davies, G. J., Walton, P. H., Johansen, K. S., Hodgson, K. O., Hedman, B., and Solomon, E. I. (2014) Spectroscopic and computational insight into the activation of O₂ by the mononuclear Cu center in polysaccharide monooxygenases, Proc. Natl. Acad. Sci.111, 8797.
Vaaje-Kolstad, G., Westereng, B., Horn, S. J., Liu, Z., Zhai, H., Sørlie, M., and Eijsink, V. G. H. (2010) An Oxidative Enzyme Boosting the Enzymatic Conversion of Recalcitrant Polysaccharides, Science330, 219-222.
Courtade, G., Ciano, L., Paradisi, A., Lindley, P. J., Forsberg, Z., Sørlie, M., Wimmer, R., Davies, G. J., Eijsink, V. G. H., Walton, P. H., and Aachmann, F. L. (2020) Mechanistic basis of substrate-O(2) coupling within a chitin-active lytic polysaccharide monooxygenase: An integrated NMR/EPR study, Proc. Natl. Acad. Sci.117, 19178-19189.
Forsberg, Z., Sørlie, M., Petrović, D., Courtade, G., Aachmann, F. L., Vaaje-Kolstad, G., Bissaro, B., Røhr, Å. K., and Eijsink, V. G. H. (2019) Polysaccharide degradation by lytic polysaccharide monooxygenases, Curr. Opin. Struct. Biol.59, 54-64.
Bissaro, B., Røhr, Å. K., Müller, G., Chylenski, P., Skaugen, M., Forsberg, Z., Horn, S. J., Vaaje-Kolstad, G., and Eijsink, V. G. (2017) Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2, Nat. Chem. Biol.13, 1123.
Bissaro, B., Streit, B., Isaksen, I., Eijsink, V. G. H., Beckham, G. T., DuBois, J. L., and Røhr, Å. K. (2020) Molecular mechanism of the chitinolytic peroxygenase reaction, Proc. Natl. Acad. Sci.117, 1504-1513.
Hangasky, J. A., Iavarone, A. T., and Marletta, M. A. (2018) Reactivity of O2 versus H2O2 with polysaccharide monooxygenases, Proc. Natl. Acad. Sci.115, 4915-4920.
Jones, S. M., Transue, W. J., Meier, K. K., Kelemen, B., and Solomon, E. I. (2020) Kinetic analysis of amino acid radicals formed in H2O2 -driven Cu(I) LPMO reoxidation implicates dominant homolytic reactivity, Proc. Natl. Acad. Sci.117, 11916-11922.
Paradisi, A., Johnston, E. M., Tovborg, M., Nicoll, C. R., Ciano, L., Dowle, A., McMaster, J., Hancock, Y., Davies, G. J., and Walton, P. H. (2019) Formation of a Copper (II)–Tyrosyl Complex at the Active Site of Lytic Polysaccharide Monooxygenases Following Oxidation by H2O2, J. Am. Chem. Soc.141, 18585-18599.
Wang, B., Wang, Z., Davies, G. J., Walton, P. H., and Rovira, C. (2020) Activation of O2 and H2O2 by Lytic Polysaccharide Monooxygenases, ACS Catal.10, 12760-12769.
Balasubramanian, R., Smith, S. M., Rawat, S., Yatsunyk, L. A., Stemmler, T. L., and Rosenzweig, A. C. (2010) Oxidation of methane by a biological dicopper centre, Nature465, 115-119.
Hakemian, A. S., and Rosenzweig, A. C. (2007) The biochemistry of methane oxidation, Annu. Rev. Biochem.76, 223-241.
Smith, S. M., Rawat, S., Telser, J., Hoffman, B. M., Stemmler, T. L., and Rosenzweig, A. C. (2011) Crystal structure and characterization of particulate methane monooxygenase from Methylocystis species strain M, Biochemistry50, 10231-10240.
Ross, M. O., MacMillan, F., Wang, J., Nisthal, A., Lawton, T. J., Olafson, B. D., Mayo, S. L., Rosenzweig, A. C., and Hoffman, B. M. (2019) Particulate methane monooxygenase contains only mononuclear copper centers, Science364, 566-570.
Tucci, F. J., Jodts, R. J., Hoffman, B. M., and Rosenzweig, A. C. (2023) Product analogue binding identifies the copper active site of particulate methane monooxygenase, Nat. Catal.6, 1194-1204.
Koo, C. W., Tucci, F. J., He, Y., and Rosenzweig, A. C. (2022) Recovery of particulate methane monooxygenase structure and activity in a lipid bilayer, Science375, 1287-1291.
Liew, E. F., Tong, D., Coleman, N. V., and Holmes, A. J. (2014) Mutagenesis of the hydrocarbon monooxygenase indicates a metal centre in subunit-C, and not subunit-B, is essential for copper-containing membrane monooxygenase activity, Microbiology160, 1267-1277.
Kim, B., Brueggemeyer, M. T., Transue, W. J., Park, Y., Cho, J., Siegler, M. A., Solomon, E. I., and Karlin, K. D. (2023) Fenton-like Chemistry by a Copper(I) Complex and H2O2 Relevant to Enzyme Peroxygenase C–H Hydroxylation, J. Am. Chem. Soc.145, 11735-11744.
Kim, S., Ginsbach, J. W., Lee, J. Y., Peterson, R. L., Liu, J. J., Siegler, M. A., Sarjeant, A. A., Solomon, E. I., and Karlin, K. D. (2015) Amine oxidative N-dealkylation via cupric hydroperoxide Cu-OOH homolytic cleavage followed by site-specific fenton chemistry, J. Am. Chem. Soc.137, 2867-2874.
Kakuda, S., Peterson, R. L., Ohkubo, K., Karlin, K. D., and Fukuzumi, S. (2013) Enhanced catalytic four-electron dioxygen (O2) and two-electron hydrogen peroxide (H2O2) reduction with a copper (II) complex possessing a pendant ligand pivalamido group, J. Am. Chem. Soc.135, 6513-6522.
Maiti, D., Narducci Sarjeant, A. A., and Karlin, K. D. (2007) Copper (II)− Hydroperoxo Complex Induced Oxidative N-Dealkylation Chemistry, J. Am. Chem. Soc.129, 6720-6721.
Yu, F., Cangelosi, V. M., Zastrow, M. L., Tegoni, M., Plegaria, J. S., Tebo, A. G., Mocny, C. S., Ruckthong, L., Qayyum, H., and Pecoraro, V. L. (2014) Protein design: toward functional metalloenzymes, Chem. Rev.114, 3495-3578.
Korendovych, I. V., and DeGrado, W. F. (2020) De novo protein design, a retrospective, Q. Rev. Biophys.53, E3.
Lombardi, A., Pirro, F., Maglio, O., Chino, M., and DeGrado, W. F. (2019) De Novo Design of Four-Helix Bundle Metalloproteins: One Scaffold, Diverse Reactivities, Acc. Chem. Res.52, 1148-1159.
Beesley, J. L., and Woolfson, D. N. (2019) The de novo design of α-helical peptides for supramolecular self-assembly, Curr. Opin. Biotech.58, 175-182.
Fletcher, J. M., Boyle, A. L., Bruning, M., Bartlett, G. J., Vincent, T. L., Zaccai, N. R., Armstrong, C. T., Bromley, E. H. C., Booth, P. J., Brady, R. L., Thomson, A. R., and Woolfson, D. N. (2012) A Basis Set of de Novo Coiled-Coil Peptide Oligomers for Rational Protein Design and Synthetic Biology, ACS Synth. Biol.1, 240-250.
Woolfson, D. N. (2021) A Brief History of De Novo Protein Design: Minimal, Rational, and Computational, J. Mol. Biol., 167160.
Tegoni, M., Yu, F., Bersellini, M., Penner-Hahn, J. E., and Pecoraro, V. L. (2012) Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils, Proc. Natl. Acad. Sci.109, 21234-21239.
Zastrow, M. L., Peacock, A. F. A., Stuckey, J. A., and Pecoraro, V. L. (2012) Hydrolytic catalysis and structural stabilization in a designed metalloprotein, Nat. Chem.4, 118-123.
Faiella, M., Andreozzi, C., De Rosales, R. T. M., Pavone, V., Maglio, O., Nastri, F., DeGrado, W. F., and Lombardi, A. (2009) An artificial di-iron oxo-protein with phenol oxidase activity, Nat. Chem. Biol.5, 882-884.
Reig, A. J., Pires, M. M., Snyder, R. A., Wu, Y., Jo, H., Kulp, D. W., Butch, S. E., Calhoun, J. R., Szyperski, T., Solomon, E. I., and DeGrado, W. F. (2012) Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins, Nat. Chem.4, 900-906.
Koebke, K. J., Yu, F., Salerno, E., Van Stappen, C., Tebo, A. G., Penner-Hahn, J. E., and Pecoraro, V. L. (2018) Modifying the Steric Properties in the Second Coordination Sphere of Designed Peptides Leads to Enhancement of Nitrite Reductase Activity, Angew. Chem. Int. Ed.57, 3954-3957.
Pirro, F., La Gatta, S., Arrigoni, F., Famulari, A., Maglio, O., Del Vecchio, P., Chiesa, M., De Gioia, L., Bertini, L., and Chino, M. (2023) A De Novo‐Designed Type 3 Copper Protein Tunes Catechol Substrate Recognition and Reactivity, Angew. Chem. Int. Ed.62, e202211552.
Mitra, S., Prakash, D., Rajabimoghadam, K., Wawrzak, Z., Prasad, P., Wu, T., Misra, S., Sharp, J., Garcia-Bosch, I., and Chakraborty, S. (2021) De Novo Design of a Self-Assembled Artificial Copper Peptide (ArCuP) that Activates and Reduces Peroxide, ACS Catal.11, 10267-10278.
Prakash, D., Mitra, S., Murphy, M., and Chakraborty, S. (2022) Oxidation and Peroxygenation of C–H Bonds by Artificial Cu Peptides (ArCuPs): Improved Catalysis via Selective Outer Sphere Modifications, ACS Catal.12, 8341-8351.
Malayam Parambath, S., Williams, A. E., Hunt, L. A., Selvan, D., Hammer, N. I., and Chakraborty, S. (2021) A De Novo-Designed Artificial Metallopeptide Hydrogenase: Insights into Photochemical Processes and the Role of Protonated Cys, ChemSusChem14, 2237-2246.
Prasad, P., Hunt, L. A., Pall, A. E., Ranasinghe, M., Williams, A. E., Stemmler, T. L., Demeler, B., Hammer, N. I., and Chakraborty, S. (2023) Photocatalytic Hydrogen Evolution by a De Novo Designed Metalloprotein that Undergoes Ni-Mediated Oligomerization Shift, Chem. Eur. J.29, e202202902.
Harbury, P. B., Zhang, T., Kim, P. S., and Alber, T. (1993) A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants, Science262, 1401-1407.
Ohta, T., Tachiyama, T., Yoshizawa, K., and Yamabe, T. (2000) Synthesis, structure, and catalytic function of a disulfide-bridged dicopper(I) complex, Tet. Lett.41, 2581-2585.
Osako, T., Nagatomo, S., Tachi, Y., Kitagawa, T., and Itoh, S. (2002) Low-Temperature Stopped-Flow Studies on the Reactions of Copper(II) Complexes and H2O2: The First Detection of a Mononuclear Copper(II)–Peroxo Intermediate, Angew. Chem. Int. Ed.41, 4325-4328.
Fujii, T., Naito, A., Yamaguchi, S., Wada, A., Funahashi, Y., Jitsukawa, K., Nagatomo, S., Kitagawa, T., and Masuda, H. (2003) Construction of a square-planar hydroperoxo-copper(ii) complex inducing a higher catalytic reactivity, Chem. Commun., 2700-2701.
Kim, S., Saracini, C., Siegler, M. A., Drichko, N., and Karlin, K. D. (2012) Coordination chemistry and reactivity of a cupric hydroperoxide species featuring a proximal H-bonding substituent, Inorg. Chem.51, 12603-12605.
Elwell, C. E., Gagnon, N. L., Neisen, B. D., Dhar, D., Spaeth, A. D., Yee, G. M., and Tolman, W. B. (2017) Copper–Oxygen Complexes Revisited: Structures, Spectroscopy, and Reactivity, Chem. Rev.117, 2059-2107.
Liu, Y., Harnden, K. A., Van Stappen, C., Dikanov, S. A., and Lu, Y. (2023) A designed Copper Histidine-brace enzyme for oxidative depolymerization of polysaccharides as a model of lytic polysaccharide monooxygenase, Proc. Natl. Acad. Sci.120, e2308286120.
Hirst, J. (2006) Elucidating the mechanisms of coupled electron transfer and catalytic reactions by protein film voltammetry, Biochim. Biophys. Acta1757, 225-239.
Marcus, R. A. (1964) Chemical and Electrochemical Electron-Transfer Theory, Ann. Rev. Phys. Chem.15, 155-196.
Marcus, R. A. (1956) On the Theory of Oxidation‐Reduction Reactions Involving Electron Transfer. I, J. Chem. Phys.24, 966-978.
Marcus, R. A., and Sutin, N. (1985) Electron transfers in chemistry and biology, Biochim. Biophys. Acta811, 265-322.
Di Bilio, A. J., Hill, M. G., Bonander, N., Karlsson, B. G., Villahermosa, R. M., Malmström, B. G., Winkler, J. R., and Gray, H. B. (1997) Reorganization Energy of Blue Copper: Effects of Temperature and Driving Force on the Rates of Electron Transfer in Ruthenium- and Osmium-Modified Azurins, J. Am. Chem. Soc.119, 9921-9922.
Farver, O., and Pecht, I. (2011) Electron transfer in blue copper proteins, Coord. Chem. Rev.255, 757-773.
Murgida, D. H., and Hildebrandt, P. (2002) Electrostatic-Field Dependent Activation Energies Modulate Electron Transfer of Cytochrome c, J. Phys. Chem. B106, 12814-12819.
Chattopadhyay, S., Mukherjee, M., Kandemir, B., Bowman, S. E. J., Bren, K. L., and Dey, A. (2021) Contributions to cytochrome c inner- and outer-sphere reorganization energy, Chem. Sci.12, 11894-11913.
Chidsey, C. E. D. (1991) Free Energy and Temperature Dependence of Electron Transfer at the Metal-Electrolyte Interface, Science251, 919-922.
Song, S., Clark, R. A., Bowden, E. F., and Tarlov, M. J. (1993) Characterization of cytochrome c/alkanethiolate structures prepared by self-assembly on gold, J. Phys. Chem.97, 6564-6572.
Nazemi, Z., Prasad, P., and Chakraborty, S. (2020) Kinetics of Oxygen Reduction by a Beta Barrel Heme Protein on Hyrid Bioelectrodes, ChemElectroChem7, 1029-1037.
Park, W.-c., and Hong, H.-G. (2006) Determination of Reorganization Energy from the Temperature Dependence of Electron Transfer Rate Constant for Hydroquinone-tethered Self-assembled Monolayers (SAMs), Bull. Korean Chem. Soc.27, 381-385.
Lee, C. W., and Anson, F. C. (1983) Recalculation of the rate of electron exchange between copper (II) and copper (I) in their 1, 10-phenanthroline and 2, 2'-bipyridine complexes in aqueous media, J. Phys. Chem.87, 3360-3362.
Winkler, J. R., Wittung-Stafshede, P., Leckner, J., Malmström, B. G., and Gray, H. B. (1997) Effects of folding on metalloprotein active sites, Proc. Natl. Acad. Sci.94, 4246-4249.
Margalit, R., Kostić, N. M., Che, C. M., Blair, D. F., Chiang, H. J., Pecht, I., Shelton, J. B., Shelton, J. R., Schroeder, W. A., and Gray, H. B. (1984) Preparation and characterization of pentaammineruthenium-(histidine-83)azurin: thermodynamics of intramolecular electron transfer from ruthenium to copper, Proc. Natl. Acad. Sci.81, 6554-6558.
Di Bilio, A. J., Dennison, C., Gray, H. B., Ramirez, B. E., Sykes, A. G., and Winkler, J. R. (1998) Electron transfer in ruthenium-modified plastocyanin, J. Am. Chem. Soc.120, 7551-7556.
Farver, O., Hosseinzadeh, P., Marshall, N. M., Wherland, S., Lu, Y., and Pecht, I. (2015) Long-Range Electron Transfer in Engineered Azurins Exhibits Marcus Inverted Region Behavior, J. Phys. Chem. Lett.6, 100-105.
Farver, O., Marshall, N. M., Wherland, S., Lu, Y., and Pecht, I. (2013) Designed azurins show lower reorganization free energies for intraprotein electron transfer, Proc. Natl. Acad. Sci.110, 10536-10540.
Farver, O., Lu, Y., Ang, M. C., and Pecht, I. (1999) Enhanced rate of intramolecular electron transfer in an engineered purple Cu_A azurin, Proc. Natl. Acad. Sci.96, 899-902.
Farver, O., Eady, R. R., and Pecht, I. (2004) Reorganization Energies of the Individual Copper Centers in Dissimilatory Nitrite Reductases: Modulation and Control of Internal Electron Transfer, J. Phys. Chem. A108, 9005-9007.
Lancaster, K. M., Farver, O., Wherland, S., Crane, E. J., Richards, J. H., Pecht, I., and Gray, H. B. (2011) Electron Transfer Reactivity of Type Zero Pseudomonas aeruginosa Azurin, J. Am. Chem. Soc.133, 4865-4873.
Lancaster, K. M., George, S. D., Yokoyama, K., Richards, J. H., and Gray, H. B. (2009) Type-zero copper proteins, Nat. Chem.1, 711-715.
Balland, V., Hureau, C., and Savéant, J.-M. (2010) Electrochemical and homogeneous electron transfers to the Alzheimer amyloid-β copper complex follow a preorganization mechanism, Proc. Natl. Acad. Sci.107, 17113-17118.
Zerk, T. J., Saouma, C. T., Mayer, J. M., and Tolman, W. B. (2019) Low Reorganization Energy for Electron Self-Exchange by a Formally Copper(III,II) Redox Couple, Inorg. Chem.58, 14151-14158.
Cao, R., Saracini, C., Ginsbach, J. W., Kieber-Emmons, M. T., Siegler, M. A., Solomon, E. I., Fukuzumi, S., and Karlin, K. D. (2016) Peroxo and Superoxo Moieties Bound to Copper Ion: Electron-Transfer Equilibrium with a Small Reorganization Energy, J. Am. Chem. Soc.138, 7055-7066.
Farver, O., Eady, R. R., Sawers, G., Prudêncio, M., and Pecht, I. (2004) Met144Ala mutation of the copper-containing nitrite reductase from Alcaligenes xylosoxidans reverses the intramolecular electron transfer, FEBS Lett.561, 173-176.
Cascella, M., Magistrato, A., Tavernelli, I., Carloni, P., and Rothlisberger, U. (2006) Role of protein frame and solvent for the redox properties of azurin from Pseudomonas aeruginosa, Proc. Natl. Acad. Sci.103, 19641-19646.
Kabsch, W. (2010) XDS, Acta Crystallogr D Biol Crystallogr66, 125-132.
Evans, P. R., and Murshudov, G. N. (2013) How good are my data and what is the resolution?, Acta Crystallogr. D Biol. Crystallogr. 69, 1204-1214.
Zwart, P. H., Grosse-Kunstleve, R. W., and Adams, P. D. (2005) phenix.xtriage, CCP4 Newsl. 43,contribution 7.
McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic software, J. Appl. Crystallogr.40, 658-674.
Potterton, L., Agirre, J., Ballard, C., Cowtan, K., Dodson, E., Evans, P. R., Jenkins, H. T., Keegan, R., Krissinel, E., and Stevenson, K. (2018) CCP4i2: the new graphical user interface to the CCP4 program suite, Acta Crystallogr. D Biol. Crystallogr. 74, 68-84.
Zaccai, N. R., Chi, B., Thomson, A. R., Boyle, A. L., Bartlett, G. J., Bruning, M., Linden, N., Sessions, R. B., Booth, P. J., Brady, R. L., and Woolfson, D. N. (2011) A de novo peptide hexamer with a mutable channel, Nat. Chem. Biol.7, 935-941.
Cowtan, K. (2006) The Buccaneer software for automated model building. 1. Tracing protein chains, Acta Crystallogr. D Biol. Crystallogr. 62, 1002-1011.
Afonine, P. V., Grosse-Kunstleve, R. W., Echols, N., Headd, J. J., Moriarty, N. W., Mustyakimov, M., Terwilliger, T. C., Urzhumtsev, A., Zwart, P. H., and Adams, P. D. (2012) Towards automated crystallographic structure refinement with phenix. refine, Acta Crystallogr. D Biol. Crystallogr. 68, 352-367.
Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics, Acta Crystallogr. D Biol. Crystallogr. 60, 2126-2132.
Murshudov, G. N., Skubak, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F., and Vagin, A. A. (2011) REFMAC5 for the refinement of macromolecular crystal structures, Acta Crystallogr. D Biol. Crystallogr. 67, 355-367.
McNicholas, S., Potterton, E., Wilson, K. S., and Noble, M. E. M. (2011) Presenting your structures: the CCP4mg molecular-graphics software, Acta Crystallogr. D Biol. Crystallogr.67, 386-394.
Stoll, S., and Schweiger, A. (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR, J. Magn. Reson.178, 42-55.

Scheme 1 is available in the Supplementary Files section.

There is NO Competing Interest.

CuRESupportingInformation.pdf
Supporting Information: X-ray data collection and refinement statistics, LSVs, electrolysis, K_M determination, and GC traces for 3SCC and 4SCC with benzyl alcohol, EPR of the ArCuPs in the absence and presence of oxidants, reduction and reoxidation monitored by Trp emission, CuI+H2O2àCuII-OOH reaction scheme, overlay of 4SCC and K6E/E8K-4SCC Cu site, detailed CV, RDE, CA data and the associated data tables, Koutecký-Levich, Arrhenius, and ln(i) vs. t plots, calculation of k_ET with O₂.
GraphicalAbstract.png
Scheme1.png
Scheme 1. H₂O₂ vs. O₂ activation pathways delineating the production of various Cu^II-oxygen species.

Download PDF

Version 1

posted

You are reading this latest preprint version

Controlling Reactivity and Electron Transfer in De Novo Designed Artificial Cu Proteins by Systematic Primary, Secondary, and Outer Sphere Modulation

Status:

Version 1

Abstract

Figures

1. INTRODUCTION

2. RESULTS AND DISCUSSION

3. SUMMARY AND CONCLUSIONS

4. EXPERIMENTAL METHODS

Declarations

References

Scheme

Additional Declarations

Supplementary Files

Status:

Version 1