Effects of l-ascorbic acid (C6H8O6: Vit-C) on collagen amino acids: DFT study

Vitamin C plays a very important role in the repair of connective tissue, especially for sports whose training causes the most damage to this tissue. Therefore, many people believe that L-ascorbic acid (C6H8O6: vitamin C) reduces the recovery time between sports exercises. The most abundant form of structural protein in the body is collagen. Collagen is characterized by a high concentration of the three amino acids glycine (Gly), proline (Pro), and hydroxyproline (Hyp), which creates its characteristic triple helix structure. Therefore, in this study, the effect of vitamin C presence on the sequence, interaction, and orientation of amino acids for collagen formation is investigated using computational simulation. This study aimed to investigate the mechanism of action of vitamin C in terms of thermodynamics and structure of the reaction. The calculations are performed using density function theory (DFT) by the base set of B3LYP/6-311++G (p,d). The results show that the presence of vitamin C is effective in the formation of collagen protein for this interaction and the mechanism of amino acid sequence (Gly-Hyp-Pro) is better in the formation of collagen protein in the presence of vitamin C. The presence of Vit-C in the formation and direction of hydroxyproline (Hyp) causes its separation from the prolyl 5-hydroxylase enzyme. In the absence of vitamin C, the reaction stops at this stage and proline cannot be converted into hydroxyproline. The computational data shows vitamin C prevents unwanted interactions and directs amino acid reactions to repair connective tissue (collagen). Therefore, vitamin C acts as a cofactor in the Prolyl 5-Hydroxylase enzyme and causes it to convert proline to hydroxyl.


Introduction
Vitamin C is one of the antioxidants that can prevent the damage of free radicals and dangerous chemicals in the body (Gęgotek and Skrzydlewska 2022).Vitamin C deficiency has signs and symptoms that cause fatigue, and weaken the strength of bones and muscles, and the body's immune system (Carr and Maggini 2017;Carr and Rowe 2020;Moore et al. 2021;Michalczyk et al. 2020).Synthesis of collagen (a protein that is important in the formation of connective tissue and wound healing) is not possible without vitamin C. Vitamin C is not made in the body and the body is not able to store it (Doseděl et al. 2021;Shetty et al. 2021;Motgi et al. 2020).
It has been proposed that the simultaneous consumption of l-arginine and vitamin C is improved the muscular endurance of athletes and is reduced the possibility of getting injured during heavy sports training, and shortens the recovery time of the body (Taherkhani et al. 2021;Dutra et al. 2020).Because the healing of musculoskeletal tissues is dependent such as bone, tendons, and ligaments, on the capacity of collagen synthesis and its cross-linking (Alipour et al. 2022;DePhillipo et al. 2018;Shaw et al. 2017;Lis and Baar 2019), the function of vitamin C in collagen synthesis should be investigated.There are two methods of clinical and computational simulation, to determine the interactions of biochemical mechanisms.In computational simulation method investigates the main reaction, and can be removed the influence of environmental factors and side reactions.
Vitamin C is essential for the function of two enzymes required in collagen synthesis: the hydroxylase, which stabilizes the collagen molecule, and the laser hydroxylase, which enhances the collagen's structural strength.In addition to enhancing the quality of collagen fibers, vitamin C affects collagen synthesis and increases collagen production (Baroroh et al. 2021;Müssig and Graupner 2021;Nieman and Wentz 2019).
Figure 1 shows the structure of collagen tissue.In all types of collagen, the two amino acids proline and glycine combine with the help of vitamin C and make procollagen (Wang et al. 2021).A collagen molecule is a triple helix formed by three polypeptide strands called alpha peptides.The amino acids present in the structure of collagen are glycine, proline, arginine, and hydroxyproline (Gupte et al. 2021).Vitamin C (ascorbate) is an essential cofactor in hydroxylation enzymes in collagen synthesis.In the hydroxylation reaction of prolyl to prolyl hydroxylase by prolyl hydroxylase domain enzymes (HIF-PHDs), ferrous (Fe 2+ ) is converted to ferric (Fe 3+ ) iron and then to Fe 4+ , which separates prolyl hydroxylase from the enzyme and HIF-PHD is reduced by vitamin C (Hosseini and Khamesee 2021).
This study investigated the mechanism of action of vitamin C in amino acid sequence using the density functional theory (DFT) method by B3LYP/6-311++G (p, d).The DFT method is based on a theorem that Hohenberg-Kohn proved in 1964 (Zhou 2012).
This theory uses electron density instead of the wave function to calculate all the frequencies of atoms and molecules.The most common DFT method is the B3LYP function, which consists of two hybrid functions (B3) and a correlation function (LYP), which is widely used in chemical processes.The 6-311++G (p, d) basis set is a polarized basis set that is used to investigate amino acids, proteins, and organic compounds (Muzomwe et al. 2012;Mojahed et al. 2022).GAMESS program package has more ability to use such functions (Guest et al. 2005).The structural parameters and electron transfer between vitamin C and the structure of amino acids can be investigated by the calculation of HOMO (the highest occupied molecular orbital) and LUMO (the lowest unoccupied molecular orbital) energy levels.

Computational method
In this study, all calculations were performed by the Spartan program at the B3LYP computational level based on 6-311++G (p, d) and also using the B3LYP computational method, which provides a very accurate prediction for covalent interactions.The DFT computational method can strike a good balance between cost and computational results (Cooperab et al. 2010;Frau and Glossman-Mitnik 2017;Wani and Mundada 2020;Brinzei et al. 2021;Ibrahimi et al. 2020).Computational software such as the GAMESS program package is needed to solve complex mathematical equations that describe the behavior of amino acids (Ibrahimi et al. 2020).
Fig. 1 The tissue-collagen structure with HOMO and LUMO orbital level energy of glycine and proline amino acids HOMO and LUMO orbitals play a very important role in the chemical stability of a compound and its interaction (Ibrahimi et al. 2020).HOMO and LUMO orbitals indicate the ability of the compound to give electrons and the ability of the compound to accept electrons, respectively (Sudha et al. 2011).The gap energy (E g ) is the difference between HOMO and LUMO orbitals.In compounds with chemically active structures, decreases the amount of gap energy decreases, but in stable structures, the gap energy increases.
The back-donation energy (Eback-donation), electronegativity (η), and hardness (µ) of chemicals indicate an increase or decrease in the chemical potential of electrons.This chemical potential depends on the amount of charge and polarization and also plays an important role in the activity of molecules and chemical structure.The chemical softness (S) determines the degree of change in the electronic arrangement of a chemical species.Compounds that have a softer structure are more chemically active.Electrophoresis (ω) represents whether a compound is more nucleophilic or electrophilic, where decreased ω represents more nucleophilic and increased ω represents more electrophilic compounds.These parameters are calculated according to the following equations (Eddy 2011;Kamada et al. 2016;Almutairi et al. 2018;Subramanian et al. 2022): (1) This research aims to investigate the effect of vitamin C in converting proline (Pro) amino acid to hydroxyproline (Hyp) to form an alpha-helix in collagen protein and investigate the direction and sequence of amino acids in collagen synthesis.

Results and discussion
Collagen proteins have glycine (Gly)-hydroxyproline (Hyp(X))-proline (Pro(Y)) sequences.The Gly-X-Y sequence allows the collagen triple helix to form.The small side chain of Glycine and the special bonding angle of Proline (hydroxyproline) stabilizes the ternary helix.Vitamin C is involved in the conversion of proline to hydroxyproline (Fig. 2) (Smirnova et al. 2012).
The prolyl 5-hydroxylase enzyme hydroxylates the proteins in the sequence of Gly-Pro-Pro and converts it to 5-hydroxyproline, thus forming the Gly-Hyp-Pro sequence (Fig. 3) (Smirnova et al. 2012).In addition to hydroxylation reactions, the formation of a stable ternary helix requires glycosylation to occur.This reaction is performed in the presence and absence of vitamin C (steps A and B in Fig. 3).
The presence of vitamin C activates the catalytic cycle of hypoxia-inducible factor prolyl hydroxylase domain enzymes (HIF-PHDs) and lysyl hydroxylase and increases the stability of alpha chains.Figures 2 and 3 show the conversion of (4) Fig. 2 The hydroxylation of the proteins in the Gly-Pro-Pro sequence to Gly-Hyp-Pro proline to 5-hydroxyproline.In the first steps (I-A and I-B), proline approaches HIF, which causes the release of a water molecule.The release of water creates the conditions for the binding and activation of oxygen (Fe 2+ → Fe 3+ ).Absorbed oxygen attacks the alpha-ketoglutarate carbonyl ketone and causes the release of CO 2 (steps II-A and II-B) and increases iron oxidation (Fe 3+ → Fe 4+ ).By absorbing a hydrogen atom from proline (Fe 4+ → Fe 3+ ), i.e. oxo-ferryl (Fe (IV) = O) converts proline into prolyl radical (steps IV-A and IV-B).If vitamin C is limited, the reaction does not progress but stops at stage A; however, if vitamin C is present, it progresses to stage B where it is converted into a dehydroascorbate prolyl radical by absorbing a hydroxyl group, converting Fe 3+ → Fe 2+ , and producing prolyl hydroxylase.
Structural and thermodynamic parameters of all stages and interactions are investigated and evaluated by their calculation by B3LYP/6-311++G (p, d) method.First, the geometric structures of all compounds used in the reactions shown in Fig. 3 are optimized by the computational method (Figs. 1, 4).The thermodynamic and structural parameters for each compound are shown in Table 1.
To investigate the effect of vitamin C, the calculations in its presence and absence are simulated, and their thermodynamic and structural parameters are calculated.
The chemical activity of compounds is investigated using structural parameters and calculating the energy level of HOMO and LUMO orbitals.As stated above in the calculations section, the higher the chemical hardness of the compound has the more stable in its structure and is been the less it tends to interact.Therefore, compounds with a softer chemical structure have more chemical activity.Table 1 shows, the structure softness (S) of proline and 2-oxoglutarate is equal to 0.077 and 0.194 eV, and the chemical hardness (µ) of their structure is equal to 6.49 and 2.57 eV, respectively.Therefore, the 2-oxoglutarate tendency to interact and release carbon dioxide gas and oxidize iron is greater than that of proline.The thermodynamic properties of the 2-oxoglutarate show the high chemical activity of this compound.
In step IV-B (Fig. 3), the catalyzed hydroxylation of HIF-PHDs interacts with Pro, and it converts to Hyp (step V-B).Thermodynamic data of Pro and Hyp structures indicate the tendency of proline amino acid for hydroxide and convert it to Hyp, which is a spontaneous reaction.The role of HIF-PHDs in this reaction is to orient and reduce proline.The geometric structure of proline hydroxide is harder (µ = 7.04 eV and S = 0.071 eV) and thus less reactive than proline (µ = 6.49eV and S = 0.077 eV).The mechanism of conversion of Pro to Hyp is simulated after to optimize the geometric structure of the compounds.Thermodynamic and structural data are calculated and shown in Tables 2 and 3 The electrical structure data of step (III) show that with the release of carbon dioxide, the structure of the complex becomes softer (S III ≈ 0.48 eV), the energy gap decreases (E g = 2.06 eV), and the structure tends to interact with Pro.In stages II and III, the presence or absence of vitamin C did not affect the gap energy.
In step (IV), the electron exchange between HIF-PHDs and proline radical takes place, which leads to a significant increase in the dipole moment and the chemical hardness of this step compared to step (III).This increase is even greater in the presence of Vit-C is then more (μ IV-A = 1.24 and μ IV-B = 6.19 eV).In the absence of vitamin C, Hyp cannot separate from the radical of HIF-PHD to create a sequence and create prolyl hydroxylase but instead stops at step 4.
However, the conversion of Vit-C to dehydroascorbate in step V allows the separation of Hyp.Step (V-B) Fig. 4 The HOMO and LUMO orbital level energy of Gly-Hyp-Pro, 2-oxoglutarate, and Succinate exhibits the lowest dipole moment, a decrease in softness (S V-B = 0.13 eV).With the introduction of Hyp into the environment, conditions are created for the production of the Gly-Hyp-Pro sequence.With the separation of Hyp in step (V-B), the conditions for succinate conversion are provided.In step (VI-B), the succinate is transformed into Calculating the total energy of each step and having the structural energy of each compound can calculate and investigate the adsorption energy.The absorption between the compounds is based on the absorption force between the absorption and absorbent molecules that can be investigated by calculating the adsorption energy.The adsorption energy (E ad ) calculated: In this equation, E c is the total energy calculated for each step.The E x , E y , and so on are the energy of other compounds in each step.Figure 5 shows the adsorption energy of compounds in the mechanism in the presence and absence of vitamin C. The changes in its E ad are very regular, and in steps I to IV, almost every path has the same adsorption energy and changes in other thermodynamic parameters.In stage III, both pathways are observed significantly in the adsorption energy, as carbon dioxide is out of interaction at this step and the electron exchange between them occurs.In the presence of vitamin C, the absorption energy is higher.With a slight decrease of pH in the environment, spontaneous absorption occurs and the reaction is performed more rapidly.
Prolyl hydroxylases participate in two catalytic reactions: (i) the hydroxylation of proline is decarboxylated to 2-oxoglutarate, and one molecule of oxygen is consumed.This reaction is independent of ascorbate, the calculated data for I-A to IV-A and I-B to IV-b showed that the reaction is better in the presence of vitamin C; (ii) Decarboxylation of 2-oxoglutarate is catalyzed by the enzyme only in the presence of ascorbate but still requires molecular oxygen.
The results show that vitamin C increases the activity of hydroxylase due to the specific reduction of Fe 3+ to Fe 2+ attached to the enzyme and during the reaction of hydroxylase.Vitamin C is not stoichiometrically consumed, so it acts as a cofactor.According to the simulation mechanism in Fig. 5, the function of vitamin C in this mechanism can be attributed to an electron donor.
Vitamin C (ascorbate) has two ionizable hydroxyl groups, which means that at physiological pH, it exists as an ascorbate monoanion (Fig. 6).It easily undergoes two  consecutive, reversible, and one-electron oxidations, which lead to ascorbate and dehydroascorbate radicals.
The ascorbate exists as ascorbate monoanion and can undergo two consecutive, reversible, and one-electron oxidations to produce ascorbate and dehydroascorbate radicals.The calculated results in Table 4 show that ascorbate is an excellent antioxidant, both thermodynamically and structurally.
The dipole moment is related to electronic structure and geometry, as the structural symmetry of the compound decreases, the dipole moment increases.Therefore, the data show that the ascorbate radical structure (R-III) has low structural symmetry.While dehydroascorbate (R-IV), this compound has the most structural symmetry (DM = 18.39D).Stable structures have more gap energy (Eg R-IV = 11.00 eV), which means that the structure of the compound is harder and its chemical softness is less.Vitamin C, as a cofactor, has a high tendency to transfer electrons, and by changing its structure, it causes the separation of Hyp from the enzyme.
The study of the mechanism of conversion of Pro to Hyp and obtaining its thermodynamic data in the presence and absence of vitamin C has been performed for the first time by the DFT method, and the obtained results show a very good agreement with the experimental data.Singh et al. (Singh et al. 2020) investigated the effect of oxygenglucose on performance Hypoxia-inducible factor (HIF) induce prolyl hydroxylase inhibitors in rats.Kuiper et al. (Kuiper et al. 2014) showed that ascorbate inhibits HIF-1 activity.Raakhuis (Braakhuis 2012) investigated the effect of vitamin C on the performance of athletes, showing that vitamin C is an essential component of the diet and may reduce the adverse effects of exercise-induced reactive oxygen species, including muscle damage, immune system dysfunction, and fatigue.These findings are consistent with our results showing that vit C plays an essential role in the healing of connective tissue and a cofactor is effective for the orientation of hydroxylase and in the orientation of amino acids and their sequences.Therefore, our model together with previous experimental evidence suggests that vit C may increase collagen synthesis and reduce recovery time following musculoskeletal injury.

Conclusion
Applying computational methods in biochemistry, pharmaceutical, medical, and other complex reactions can determine the impact of materials and mechanisms on their performance (Sherafatizangeneh et al. 2022).
In this study, the role of vitamin C in the sequence of amino acids for collagen connective tissue was simulated, and thermodynamic and structural parameters were calculated using DFT calculation methods.The data show, that vitamin C plays an essential role in the healing of connective tissue and is a cofactor for iron-dependent enzymes and 2-oxoglutarate dioxygenase.The enzyme prolyl 5-hydroxylase (HIF) creates a hydroxyl group (OH) to the gamma carbon atom in proline and converts it to hydroxyproline, which requires ascorbate as a cofactor for its activity.The results show under conditions of Vit-C deficiency or absence, HIF loses its activity, collagen cannot sufficiently cross-link, and connective tissues can deteriorate.

Fig. 3
Fig. 3 The side-reaction of HIF-PHDs in the absent (step-A) and presence of vitamin C (step-B), and oxygen (steps I-A to IV-A and I-B to VI-B, see explanation in text) . The values of reaction heat (H o ) and Gibbs free energy (G o ) indicate that the reactions are spontaneous.Steps (I-A and I-B) are the interaction of the hydroxylation of HIF-PHDs in the presence of proline with 2-oxoglutarate, which absorbs oxygen and loses the water molecule (steps II-A and II-B).Computational data show that in the acidic environment in the presence of vitamin C (step-B), the dipole moment (DM) and electronegativity (η) of the interaction increase and the total energy shows a significant decrease in the presence of vitamin C. Additionally, the dipole moment and electronegativity of the interaction increase, and the total energy shows a significant decrease compared to the environment without vitamin C. The enthalpy difference (Δ → ) for steps A and B is − 280.02129 and − 280.18113 a.u. and Δ → is − 280.031401 and − 280.19106 a.u., respectively.Other thermodynamic parameters show that the reaction is more spontaneous in the presence of vitamin C. The gap energy decreases in the second step of the simulated mechanism (E g(II-A and II-B) = 4.36 eV), which increases the electron exchange and conductivity of the complex.In step (III), the enzyme-bound 2-oxoglutarate becomes succinate, losing a molecule of carbon dioxide and becoming a regenerator that is ready to reduce proline and the dipole moment and decreases the chemical hardness of the compound.

Fig. 5
Fig. 5 DFT calculations of the adsorption energy of reactants in all steps of the mechanism of conversion of Pro to Hyp for determining the role of vitamin C: A the absence, and B the presence of Vit-C

Fig. 6
Fig. 6 Chemical structures of ascorbate and its oxidation products

Table 1
Thermodynamic and structural parameters of the geometric structure of compounds

Table 3
Thermodynamic and structural parameters of steps I-B to VI-B in the presence of vitamin C