Concerning the Application of the Q Cycle to Electron Transport in Cyanobacteria

doi:10.21203/rs.3.rs-3463667/v1

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Concerning the Application of the Q Cycle to Electron Transport in Cyanobacteria

https://doi.org/10.21203/rs.3.rs-3463667/v1

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A working concept for the regulation of electron transport in oxygenic photosynthesis is that the electron transfer rate between the two photosystems, PSI and PSII, is governed by a ‘Q-cycle’ pathway operating in the electron transport chain which connects the two ‘reaction center’ complexes. The ‘Q-cycle’ concept was initially inferred from studies on mitochondrial electron transport. This concept has been assumed to be relevant to the electron transport pathways operating in oxygenic photosynthesis, with the majority of studies done on chloroplasts or thylakoid membranes The present study examines the existence and properties of a putative ‘Q-cycle’ in cyanobacteria. Light-induced spectral changes associated with cytochrome redox reactions in intact cells of the cyanobacterium Synechococcus sp. corresponded to the oxidation-reduction of cytochrome f. A correlated reduction of heme b₆ was, however, not observed. The absence of significant cytochrome b reduction might be considered inconsistent with the set of electron transfer events associated conceptually with a ‘Q-cycle’ model of the electron transfer events in the chain. However, because heme b₆ in the intact cyanobacteria is mostly reduced, it is not observable as a net electron acceptor of the plastoquinol or semiquinone formed by electron transfer from photosystem II. The redox environment of intact cyanobacteria in the dark resting state has an ambient potential sufficiently reducing that the ‘Q-cycle’ pathway for electron transport, well studied and characterized for function in isolated thylakoid membranes or chloroplasts, is not observed. This apparent quandary’ is a consequence of the reducing (negative potential) intracellular redox environment of cyanobacteria, which imposes a reduced state on the b-hemes, thereby preventing observation of their light-induced reduction.

Under conditions of optimum light intensity and carbon dioxide concentration, the rate-determining step of oxygenic photosynthesis via non-cyclic electron transport is electron transfer from plastoquinol (PQH₂)/semiquinone) to the high potential non-heme iron-sulfur (‘Rieske’) protein and a low potential cytochrome (cyt b₆) heme of the electron transport chain. It has been proposed that details of electron transfer in the quinol oxidation reaction are described by a ‘Q-cycle’ model, based on the realization of the ubiquinone fluidity proposed for the ubiquinol-cytochrome b reaction in mitochondria (Mitchell, 1976; Slater, 1983; Schneider et al., 1985; Trumpower, 2002), and subsequently studied in photosynthetic bacteria (Crofts and Meinhardt, 1983; Crofts et al., 1983; Joliot, P. and Joliot, A., 2002), its properties described in some detail in a textbook Cramer and Knaff (1990). Regarding application of the Q-cycle model to oxygenic photosynthesis, a goal of the present study is to provide such documentation for the redox changes associated with quinol oxidation, and the cytochrome redox changes, in a cyanobacterium (PC7002, Synechococcus sp). A feature of the present study is a comparison of these redox properties with those reported previously for plant thylakoid membranes (Cramer and Butler, 1967; Cramer and Hasan, 2016).

It is noted in the present discussion that the two trans-membrane b hemes, one on the electrochemically negative (n) and one on the positive (p), ‘stromal’ and ‘granal’ sides, respectively) in the cytochrome b₆f complex, with standard potentials -0.1 - + 0.1 V) are mostly reduced in the active growth phase of cultures of the cyanobacterium, thus defining a redox environment seemingly unfavorable for operation of a Q cycle. It was, therefore, inferred that proton translocation associated with quinol oxidation at this site might occur through a different (e. g., H⁺ pump) mechanism, involving the high potential iron-sulfur (Rieske) protein (Kramer and Crofts, 1990; Papa et al., 1983). Here it is proposed that the different results obtained in ‘flash-kinetic’ experiments with (i) isolated plant chloroplasts compared to (ii) intact photosynthetic microbial cells do not imply any difference in the pathways and mechanisms of oxygenic photosynthetic electron transport, but rather a difference in the ambient redox environment of the hydrophobically intact cell interior and the more polar environment of the redox carriers in isolated chloroplasts.

1. Growth of Cyanobacterial Cultures. Synechococcus sp. PCC 7002, wild-type, were grown in medium A (Hasan et al., 2018) under optimal growth conditions, 38°C with an incident light intensity of 100–150 micro Einsteins·m⁻²·s⁻¹ in 2% (v/v) CO₂/air. The cell titer was assayed using a Cary 3 spectrophotometer through the optical density of the cell culture measured at 660 nm relative to that at a reference wavelength of 730 nm, using a spectrophotometer with the light collection and detector geometry designed for a high efficiency of collection of light scattered from the turbid cell suspension.

2, Preparation of Spinach Chloroplast Thylakoid Membranes. Thylakoid membranes were prepared according to (Hasan and Cramer (2014); Hasan et al., 2014). Spinach leaves (15 g) were homogenized (in the presence of 100 ml of buffer A or grinding buffer at high speed for 30 seconds. The resulting mixture was passed through a cloth filter. The filtrate was then separated by centrifugation (2 min, 2,000 x g, 4^oC), and the isolated pellet resuspended in ‘buffer B’ (Cramer and Hasan, 2016) and incubated on ice for 10 min. The solution was centrifuged twice at 4000 rpm for 10 min, and the resulting sediment stored in buffer B containing protease inhibitors. Aliquots of this suspension were used to carry out determinations of chlorophyllconcentration.

3. Flash Kinetic Spectroscopy. To measure cytochrome f oxidation, wild type Synechococcus was used at a final chlorophyll concentration of 50-100 μg/ml in the presence of 0.5 mM methyl viologen (MV) as the terminal electron acceptor. Flash-induced oxidation of cytochrome f, using a triggered Xenon flash single-beam spectrometer (Zhang et al., 2008) was measured at 554 nm relative to 540 nm as a reference wavelength, thus generating the difference absorbance signal, ΔA_554–540. An ‘action spectrum’ which describes the light-induced oxidation of cytochrome f was obtained by repeating this measurement over the wavelength range (548–560 nm) that spans the cytochrome alpha-band spectral domain. The same procedure was used to obtain an action spectrum associated with the light-induced redox change for heme b₆ reduction in spinach thylakoid membranes, with the peak of its chemical redox difference spectrum at 563 nm, using a reference wavelength of 575 nm. Duroquinol at a final concentration of 0.5 mM, and the ‘uncoupler’ gramicidin (5 μM) were added to the reaction mixture to facilitate regeneration of reduced plastoquinone and cytochrome f. The cell suspension was diluted to an optical density of 1.0 at 730 nm for spectrophotometric analysis, the output signal referenced to the pre-flash output function, and a noise suppression algorithm utilized to minimize noise in the signal and detector output.

The pattern of absorbance and resulting spectral changes associated with light- and chemically - induced changes in the oxidation state of cytochrome f has been documented in previous studies on spinach thylakoids and C. reinhardtii (Cramer and Hasan, 2016). The present study extended this analysis to the redox changes of the cytochrome b₆f complex in the cyanobacterium, Synechococcus sp. PCC 7002. The light-induced redox changes were referenced to those obtained by actinic Illumination of spinach chloroplasts, suspended at a concentration of 50-100 μg/ml chlorophyll with methyl viologen (0.5 mM), as the electron acceptor. The peak of the spectrum generated to characterize the extent of the cytochrome-specific redox changes (Cramer and Hasan, 2016; Kramer and Crofts, 1990) is close to 554 nm (Fig. 1A), the α- band (in green region of spectrum)-peak of the cytochrome f redox difference spectrum.

A spectrum of the light-induced absorbance change, an example shown in Fig. 1B, was generated from the set of individual absorbance measurements at (Fig. 1A). The peak of the difference spectrum as defined by ‘best fit’ analysis shows that the spectrum peaks at 555 nm. The difference spectrum for oxidation of heme b in the b₆f complex was centered at 563-564 nm (Fig. 1B). These spectra are consistent with the well-documented precedent for the spectrum for light-induced oxidation of cytochrome f (Cramer and Hasan, 2016; Hasan et al., 2018), and also provide a spectrophotometric reference for the additional studies reported here on the amplitude and kinetics of the light-induced redox turnover of b-cytochromes in intact cyanobacteria. The amplitude and time course of ‘dark reduction,’ the redox change in the dark to light-induced oxidation of cytochrome f in spinach thylakoid membranes, measured by the absorbance change at 554 nm relative to a reference change measured at 540 nm, is shown (Fig. 2A). The first 40 ms in the display show a baseline which displays (i) the background noise in the measurement followed by (ii) a spike arising from leak into the detector of the light flash at approximately 40 ms into the recording In the absence or presence of a sample. The amplitude of the effective absorbance change associated with a leak of the relatively intense actinic light flash, if displayed alone, would decrease immediately after termination of the light flash. However, a relatively slow decrease in absorbance is seen with a half-life of 8-10 msec which arises from the absorbance change associated with (i) the light-induced oxidation, the detailed time course not resolved here, and (ii) the subsequent ‘dark reduction’ of cytochrome f after termination of the light flash. The time course of the initial light-induced oxidation of the cytochrome was not resolved in these measurements.

Under the same experimental conditions, similar measurements were done at a measuring light wavelength of 563 nm to determine the spectral properties of the heme b₆ redox change (Fig. 2B). The reaction medium contained duroquinol as a reductant of the quinone pool and gramicidin as an uncoupler of electron transfer. A ‘slow’ oxidation of heme b, consistent with prediction and previous observations was observed. Presumably because of the difference in rateconstants for oxidation and reduction, the change in absorbance for heme b₆ is significantly smaller than that of cytochrome f. The critical experiment which describes the presence of these absorbance changes in intact cells, spectrophotometric measurement at the alpha-band absorbance peaks associated with cytochromes f and b₆, is shown (Figs. 3A, B).

1. Growth of Cyanobacterial Cultures. Synechococcus sp. PCC 7002, wild-type, were grown in medium A (Hasan et al., 2018) under optimal growth conditions, 38°C with an incident light intensity of 100–150 micro Einsteins·m^− 2·s^− 1 in 2% (v/v) CO₂/air. The cell titer was assayed using a Cary 3 spectrophotometer through the optical density of the cell culture measured at 660 nm relative to that at a reference wavelength of 730 nm, using a spectrophotometer with the light collection and detector geometry designed for a high efficiency of collection of light scattered from the turbid cell suspension.

3. Flash Kinetic Spectroscopy. To measure cytochrome f oxidation, wild type Synechococcus was used at a final chlorophyll concentration of 50–100 µg/ml in the presence of 0.5 mM methyl viologen (MV) as the terminal electron acceptor. Flash-induced oxidation of cytochrome f, using a triggered Xenon flash single-beam spectrometer (Zhang et al., 2008) was measured at 554 nm relative to 540 nm as a reference wavelength, thus generating the difference absorbance signal, ΔA_554–540. An ‘action spectrum’ which describes the light-induced oxidation of cytochrome f was obtained by repeating this measurement over the wavelength range (548–560 nm) that spans the cytochrome alpha-band spectral domain. The same procedure was used to obtain an action spectrum associated with the light-induced redox change for heme b₆ reduction in spinach thyla-

It is shown here that a major difference exists between the results of visible light flash excitation experiments on the b-hemes obtained (a) with thylakoid (e. g., spinach) membranes and (b) cyanobacteria, represented here by the studies on Synechococcus (Fig. 2B and 3B, respectively). Thus, based on a measured absorbance change of approximately 1.5 x 10^-3 for cytochrome f at 554 nm (Fig. 3A), a significant amplitude for photoreduction of heme b₆, assayed through the magnitude of the absorbance difference at 563-564 nm, was not detected. The detector of the spectrophotometric device has an intrinsic noise level of 0.5 x 10^-4 absorbance units. Thus, if there is a light-induced change in the b₆heme, it is substantially smaller than would be expected for any significant amplitude of reduction.

In summary, the ratio of the light-induced absorbance change between heme b₆and cytochrome f is significantly smaller than 0.5, partly a consequence of the structural proximity of cytochrome f to the photosystem I reaction center. These studies suggest an alternate pathway and mechanism for energy transduction linked to electron transport in the ‘main’ electron transport chain linking the two photosystems. Regarding the location of this additional site for energy transduction, It has been proposed that a significant component of the proton electrochemical potential gradient in oxygenic photosynthesis could be provided through a pathway and mechanism of proton translocation involving conformation changes of the Rieske protein present in the mitochondrial cytochrome bc₁ complex (Papa et al., 1983). The latter model combines ‘proton-motive’ catalysis by quinone bound to the cytochrome bc₁ complex, and proton conduction along intra-membrane, trans-membrane pathways in the apoproteins of the cytochrome bc₁ complex.

Acknowledgments

WACproposedthe study; AP carried out the spectrophotometric analysis as part of an undergraduate research project. The authorsthank the College of Science. Purdue University, for providing a summer research Fellowship to AP, and the Department of Biological Sciences, for providing technical support of the facilities, with no competing interests. We thank Ms. Rebecca Harding for assistance in the assembly and submission of the manuscript.

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Concerning the Application of the Q Cycle to Electron Transport in Cyanobacteria

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