Proton-Coupled Electron Transfer in Cytochrome c Oxidase: Heme a Controls the Protonation Dynamics of E286.

Coupled electron and proton transfer reactions play a key role in the mechanisms of biological energy transduction.1–3 Such reactions are also fundamental for artificial energy-related systems such as fuel cells, chemical sensors, and other electrochemical devices. Biological examples include, among others, cytochrome c oxidase,4,5 bc1 complex,6,7 and photosynthetic reaction centers.8,9 In such systems, electrons tunnel between redox cofactors of an enzyme, while the coupled protons are transferred either across a single hydrogen bond or between protonatable groups along special proton-conducting channels. In this paper general theories and models of coupled electron transfer/proton transfer (ET/PT) reactions are discussed. Pure electron transfer reactions in proteins have been thoroughly studied in the past, both experimentally10–17 and theoretically.18–25 The coupled reactions are relatively new and currently are gaining attention in the field.6,8,26–43 Two types of coupled reactions can be distinguished. In concerted electron and proton transfer reactions (denoted PCET in Refs. 29,30,43–45, although this term is also used more generally), both the ET and PT transitions occur in one step. Such concerted processes occur in reactions in which proton transfer is typically limited to one hydrogen bond; however, examples with multiple hydrogen bond rearrangements are also known.46 In sequential reactions, the transitions occur in two steps: ET/PT or PT/ET. Typically each individual step is uphill in energy, while the coupled reaction is downhill. A sequential reaction can proceed along two parallel channels: ET then PT (EP) or PT then ET (PE). In each channel the reaction involves two sequential steps: uphill activation, and then downhill reaction to the final product state. The lifetime of the activated complex is limited by the back reaction. The general formula for the rate of such reactions can be easily developed. In the context of bioenergetics issues, however, it is interesting to analyze all of the possible cases separately because each corresponds to a different mechanism: for example, an electron can go first and pull out a proton; alternatively, a proton can go first and pull out an electron; or an electron can jump back and forth between donor and acceptor and gradually pull out a proton. In enzymes involving coupled proton and electron transport, the exact mechanism of the reaction is of prime interest. First we will consider a simple four-state model of reactions where the proton moves across a single hydrogen bond; both concerted and sequential reactions will be treated. Then we will consider models for long-distance proton transfer, also denoted proton transport or proton translocation. Typically, electron transfer coupled to proton translocation in proteins involves an electron tunneling over a long distance between two redox cofactors, coupled to a proton moving along a proton conducting channel in a classical, diffusion-like random walk fashion. Again, separately the electron and proton transfer reactions are typically uphill, while the coupled reaction is downhill in energy. The schematics of this process is shown in Fig. 1. The kinetics of such reactions can be much different from those involving proton transfer across a single hydrogen bond. In this paper, we will discuss the specifics of such long-distance proton-coupled reactions. Fig. 1 Schematics of the electron transfer reaction coupled to proton translocation. In the reaction, an electron is tunneling over a long distance between two redox cofactors, O and R, and a coupled proton is transferred over a proton conducting channel. The ... Following the review of theoretical concepts, a few applications will be discussed. First the phenoxyl/phenol and benzyl/toluene self-exchange reactions will be examined. The phenoxyl/phenol reaction involves electronically nonadiabatic proton transfer and corresponds to a proton-coupled electron transfer (PCET) mechanism, whereas the benzyl/toluene reaction involves electronically adiabatic proton transfer and corresponds to a hydrogen atom transfer (HAT) mechanism. Comparison of these two systems provides insight into fundamental aspects of electron-proton interactions in these types of systems. Next a series of theoretical calculations on experimentally studied PCET reactions in solution and enzymes will be summarized, along with general predictions concerning the dependence of rates and kinetic isotope effects (the ratio of the rate constants for hydrogen and deuterium transfer) on system properties such as temperature and driving force. The final application that will be discussed is cytochrome c oxidase (CcO). CcO is the terminal component of the electron transport chain of the respiratory system in mitochondria and is one of the key enzymes responsible for energy generation in cells. The intricate correlation between the electron and proton transport via electrostatic interactions, as well as the kinetics of the coupled transitions, appear to be the basis of the pumping mechanism in this enzyme.

Review: 180 refs.

Identifying the proton loading site cluster in the ba3 cytochrome c oxidase that loads and traps protons

The electron transfer (ET) reactions in mitochondrial and bacterial respiratory chains are essential processes for energy transduction in cells. A series of ET reactions is terminated at cytochrome c oxidase (CcO), where molecular oxygen is reduced to water. Associated with the reduction of molecular oxygen, CcO functions as a proton pump across the membrane, and the proton gradient is the primary driving force for the generation of ATP. In the respiratory chain of mitochondria, the electrons to reduce molecular oxygen at CcO are donated from a small hemoprotein, cytochrome c (Cyt c), and Cyt c forms an ET complex with CcO to promote the ET reaction. While the reduction of molecular oxygen to water molecules requires four electrons, Cyt c can carry only one electron, implying that Cyt c repetitively associates with and dissociates from CcO and suggesting that the specific interprotein interactions between Cyt c and CcO to form the ET pathway from Cyt c to CcO. To identify the specific interactions for the complex formation between Cyt c and CcO and estimate the ET pathway, we have determined the interaction site of Cyt c for CcO using NMR spectroscopy1. However, the NMR measurements can be applied only for ET complexes such as oxidized Cyt c – fully oxidized CcO or reduced Cyt c – fully reduced CcO complexes, where the ET reaction does not occur. In addition, our previous NMR analysis1 identified the CcO interaction site on Cyt c, while no information about the Cyt c interaction site on CcO has been obtained. A detailed analysis of the interactions essential for the formation of the ET pathway in the ET complexes under turnover conditions, where the ET reaction from Cyt c to CcO is induced, has not yet been conducted. Based on the mutational effects on the steady state kinetics of the ET reaction and our NMR analysis of the interaction site, we examined the structure of the ET complex between Cyt c and CcO under turnover conditions using protein docking simulation2, and energetically characterized the interactions essential for complex formation and estimated the ET pathway from Cyt c to CcO. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the ET reaction to CcO. The interaction analysis using the selected Cyt c – CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable (desolvation) electrostatic interactions which were almost cancelled out by large favorable (Columbic) electrostatic interactions, but resulting in the destabilization of the complex. The residual destabilizing free energy is compensated by the van der Waals interactions mediated by hydrophobic amino acid residues to give the stabilized complex. Thus, hydrophobic interactions are the primary factors that promote complex formation between Cyt c and CcO under turnover conditions, while the change in the electrostatic destabilization free energy provides the variance of the binding free energy in the mutants. The distribution of favorable and unfavorable electrostatic interactions in the interaction site determines the orientation of the binding of Cyt c on CcO. To determine the ET pathway from Cyt c to CcO, the pathway analysis3 was applied to the predicted ET complex between Cyt c and CcO under turnover conditions. The estimated ET pathway was found to be constructed by many hydrophobic amino acid residues, including Trp104 of the subunit II of CcO, which is supposed to be the electron entry site for CcO from Cyt c. The contribution of many hydrophobic amino acid residues to formation of the ET pathway also supports the crucial role of the dehydration from hydrophobic amino acid residues associated with the complex formation between Cyt c and CcO, and suggests formation of a structure blocking water access from the bulk water phase (a “molecular breakwater”) surrounding the ET pathway across the Cyt c - CcO interface to avoid formation of hydrogen-bond mediated non-specific ET pathways. Sakamoto, K.; Kamiya, M.; Imai, M.; Shinzawa-Itoh, K.; Uchida, T.; Kawano, K.; Yoshikawa, S.; Ishimori, K., Proc. Natl. Acad. Sci. USA 2011, 108, 12271-12276.Sato, W.; Hitaoka, S.; Inoue, K.; Imai, M.; Saio, T.; Uchida, T.; Shinzawa-Itoh, K.; Yoshikawa, S.; Yoshizawa, K.; Ishimori, K., J. Biol. Chem. 2016, 291, 15320-15331.Onuchic, J. N.; Beratan, D. N.; Winkler, J. R.; Gray, H. B., Ann. Rev. Biophys. Biomol. Struct. 1992, 21, 349-377.

Proton-coupled electron transfer (PCET) plays a crucial role in many enzymatic reactions and is relevant for a variety of processes including water oxidation, nitrogen fixation, and carbon dioxide reduction. Much of the research on PCET has focused on transfers between molecules in their electronic ground states, but increasingly researchers are investigating PCET between photoexcited reactants. This Account describes recent studies of excited-state PCET with d(6) metal complexes emphasizing work performed in my laboratory. Upon photoexcitation, some complexes release an electron and a proton to benzoquinone reaction partners. Others act as combined electron-proton acceptors in the presence of phenols. As a result, we can investigate photoinduced PCET involving electron and proton transfer in a given direction, a process that resembles hydrogen-atom transfer (HAT). In other studies, the photoexcited metal complexes merely serve as electron donors or electron acceptors because the proton donating and accepting sites are located on other parts of the molecular PCET ensemble. We and others have used this multisite design to explore so-called bidirectional PCET which occurs in many enzymes. A central question in all of these studies is whether concerted proton-electron transfer (CPET) can compete kinetically with sequential electron and proton transfer steps. Short laser pulses can trigger excited-state PCET, making it possible to investigate rapid reactions. Luminescence spectroscopy is a convenient tool for monitoring PCET, but unambiguous identification of reaction products can require a combination of luminescence spectroscopy and transient absorption spectroscopy. Nevertheless, in some cases, distinguishing between PCET photoproducts and reaction products formed by simple photoinduced electron transfer (ET) (reactions that don't include proton transfer) is tricky. Some of the studies presented here deal directly with this important problem. In one case study we employed a cyclometalated iridium(III) complex. Our other studies with ruthenium(II) complexes and phenols focused on systematic variations of the reaction free energies for the CPET, ET, and proton transfer (PT) steps to explore their influence on the overall PCET reaction. Still other work with rhenium(I) complexes concentrated on the question of how the electronic structure of the metal-to-ligand charge transfer (MLCT) excited states affects PCET. We used covalent rhenium(I)-phenol dyads to explore the influence of the electron donor-electron acceptor distance on bidirectional PCET. In covalent triarylamine-Ru(bpy)₃²⁺/Os(bpy)₃²⁺-anthraquinone triads (bpy = 2,2'-bipyridine), hydrogen-bond donating solvents significantly lengthened the lifetimes of photogenerated electron/hole pairs because of hydrogen-bonding to the quinone radical anion. Until now, comparatively few researchers have investigated this variation of PCET: the strengthening of H-bonds upon photoreduction.

Complex I functions as an initial electron acceptor in aerobic respiratory chains that reduces quinone and pumps protons across a biological membrane. This remarkable charge transfer process extends ca. 300 Å and it is initiated by a poorly understood proton-coupled electron transfer (PCET) reaction between nicotinamide adenine dinucleotide (NADH) and a protein-bound flavin (FMN) cofactor. We combine here large-scale density functional theory calculations and quantum/classical models with atomistic molecular dynamics simulations to probe the energetics and dynamics of the NADH-driven PCET reaction in complex I. We find that the reaction takes place by concerted hydrogen atom (H•) transfer that couples to an electron transfer (eT) between the aromatic ring systems of the cofactors and further triggers reduction of the nearby FeS centers. In bacterial, Escherichia coli-like complex I isoforms, reduction of the N1a FeS center increases the binding affinity of the oxidized NAD+ that prevents the nucleotide from leaving prematurely. This electrostatic trapping could provide a protective gating mechanism against reactive oxygen species formation. We also find that proton transfer from the transient FMNH• to a nearby conserved glutamate (Glu97) residue favors eT from N1a onward along the FeS chain and modulates the binding of a new NADH molecule. The PCET in complex I isoforms with low-potential N1a centers is also discussed. On the basis of our combined results, we propose a putative mechanistic model for the NADH-driven proton/electron-transfer reaction in complex I.

An interaction between cytochrome a in oxidized cytochrome c oxidase (CcO) and anions has been characterized by EPR spectroscopy. Those anions that affect the EPR g = 3 signal of cytochrome a can be divided into two groups. One group consists of halides (Cl-, Br-, and I-) and induces an upfield shift of the g = 3 signal. Nitrogen-containing anions (CN-, NO2-, N3-, NO3-) are in the second group and shift the g = 3 signal downfield. The shifts in the EPR spectrum of CcO are unrelated to ligand binding to the binuclear center. The binding properties of one representative from each group, azide and chloride, were characterized in detail. The dependence of the shift on chloride concentration is consistent with a single binding site in the isolated oxidized enzyme with a Kd of approximately 3 mm. In mitochondria, the apparent Kd was found to be about four times larger than that of the isolated enzyme. The data indicate it is the chloride anion that is bound to CcO, and there is a hydrophilic size-selective access channel to this site from the cytosolic side of the mitochondrial membrane. An observed competition between azide and chloride is interpreted by azide binding to three sites: two that are apparent in the x-ray structure plus the chloride-binding site. It is suggested that either Mg2+ or Arg-438/Arg-439 is the chloride-binding site, and a mechanism for the ligand-induced shift of the g = 3 signal is proposed.

Therapeutic decisions in colorectal cancer (CRC) will be enhanced if guided by more accurate prognostic and predictive biomarkers during the progression of adenomas to CRC. Given that most CRCs develop from adenopolyps via the adenoma-carcinoma sequence, a mechanism for the inhibition of this sequence in patients with a high risk of developing CRC is a pressing need. Variants in mitochondrial (mt) protein expressions have been correlated with several clinico-pathological features of cancers as the majority of energy for tumor transformation are of mitochondrial origin. Differences in mitochondrial efficiency may be reflected as in adenoma-carcinoma sequence. Reports have shown that cytochrome c oxidase (COX) is a key player in oxidative phosphorylation and reactive oxygen species (ROS) formation. In addition ATPase subunits are also associated with ROS formation and mtDNA maintenance. Here, we specifically searched for differentially expressed ATPase and COX subunits in early adenomas of CRC tissues as compared to late stages of CRC tissues. In addition mitochondria variants were analyzed in mitochondrial encoded subunits of complexes IV and V of the electron transport chain. Direct sequencing, high resolution restriction digestion, RT-qPCR and western blot techniques were used to assess differences in colorectal tumors. Tissue samples used included early adenomas classified as tubular adenoma (TA), tubulovillous adenoma (TV), and villous adenomas (v); cancer tissues, and normal surrounding tissues. Results suggest that most variants of complex IV were found in mitochondrial encoded cytochrome c oxidase subunit III (9207-9990). Of these variants 9414delC found in 60% TA and 20% CA samples was predicted to be disease causing. Furthermore, ATPase6 variant G9055A found abundantly in TA and V samples was confirmed using high resolution restriction digestion. Expression levels of ATPase6 progressively increased from early adenomas to late stage adenomas and cytochrome c oxidase mitochondrial subunits also varied within the adenoma carcinoma sequence. Interestingly, COX subunit 4 isoform 1 protein expression decreased by five-fold in cancer samples when compared to normal tissue and by three-fold when compared to TA. Therefore, this study suggest an important role of ATPase and COX IV-1 in tumor CRC progression in respect to the impact on mitochondrial ROS production and oxidative phosphorylation regulation. Citation Format: Lashanale Wallace, Anju Cherian, Felix Aikhionbare. Differential expression of MT-ATPase and COXIV genes in colorectal adenopolyps [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2228.

There is a growing appreciation of the important role that H-bond complexes can play in the mechanism of proton-coupled electron transfer (PCET) reactions. Until relatively recently it had been thought that PCET reactions always proceeded step-wise with sequential electron and proton transfer. Much of the recent fundamental interest in PCET stems from the realization that a third option is available, concerted electron and proton transfer or CPET in which the electron and proton both move in a single kinetic step. This interest in the concerted process has increased awareness of H-bonding states in PCET, since the concerted reaction occurs within a H-bonded intermediate. However, even if the proton and electron transfer is not concerted, the H-bonded complex formed in the process of proton transfer can play an important role in the PCET mechanism if it is sufficiently long-lived.Recently we have introduced a generally useful mechanistic framework with which to include H-bonding steps within an overall PCET pathway. This scheme, which for obvious reasons we call a “wedge”, is shown in Scheme 1 for the generic 1e−, 1H+ oxidation, AH + B = A + HB+ + e−. The front face in the wedge (in bold) is the standard electron transfer/proton transfer square scheme, with the two possible electron transfer reactions on the top and bottom edges, and the two possible proton transfers on the left and right edges. However, proton transfer reactions actually go through a H-bond intermediate, so a more accurate description of the proton transfer follows the dashed lines on the triangular sides of the wedge to and from the H-bond intermediates, A-H-B or A-H-B+, which are meant to represent the thermodynamically most stable H-bond complex in each oxidation state. If the H-bonded intermediate has sufficient lifetime, then electron transfer to/from the H-bond complex is also possible, represented by the rear edge of the wedge (thin solid line). If the proton moves from being more attached to A in A-H-B to being more attached to B in A-H-B+, then E° of this reaction is that of the CPET step, if the proton doesn’t move then the E° is simply that of oxidation of the H-bond complex. Either way, it is straightforward to show that E°(A-H-B0/+) has to have a value in between E°(AH0/+) and E°(A−/0). Thus the possibility of electron transfer through the H-bond complex opens up a pathway of intermediate potential for the overall reaction AH + B = A + HB+ + e−.The usefulness of the wedge scheme is demonstrated by its ability to explain the unusual electrochemistry of the phenylenediamine-based urea, U(H)H, which we have shown undergoes a self proton transfer upon oxidation to give half equivalent of the doubly oxidized quinoidal cation and half-equivalent of the electroinactive, protonated reduced urea, Scheme 2. The reaction gives chemically irreversible voltammetry in acetonitrile as would be expected given that the quinoidal cation is harder to reduce than the initially formed radical cation. However, it gives reversible voltammetry in methylene chloride, which can be explained by the greater stability of the H-bonded intermediate in this solvent. In addition, in methylene chloride, we are able to clearly observe a concentration and scan rate dependent conversion between two different reduction pathways on the return scan. This behavior cannot be explained by a simple square scheme, but is readily explained by the wedge scheme.In this presentation, we will report recent results on the voltammetry of U(H)H in the presence of guest molecules that H-bond to the starting, reduced state. We will show that their effect on the voltammetry can be explained in terms of two interlinked wedge schemes, one representing the electron transfer / H-bonding / proton transfer reactions of U(H)H with itself and the other representing the reactions with the added guest.

The coupling of electron and proton transfer is an important controlling factor in radical proteins, such as photosystem II, ribinucleotide reductase, cytochrome oxidases, and DNA photolyase. This was investigated in model complexes in which a tyrosine or tryptophan residue was oxidized by a laser-flash generated trisbipyridine-Ru(III) moiety in an intramolecular, proton-coupled electron transfer (PCET) reaction. The PCET was found to proceed in a competition between a stepwise reaction, in which electron transfer is followed by deprotonation of the amino acid radical (ETPT), and a concerted reaction, in which both the electron and proton are transferred in a single reaction step (CEP). Moreover, we found that we could analyze the kinetic data for PCET by Marcus' theory for electron transfer. By altering the solution pH, the strength of the Ru(III) oxidant, or the identity of the amino acid, we could induce a switch between the two mechanisms and obtain quantitative data for the parameters that control which one will dominate. The characteristic pH-dependence of the CEP rate (M. Sjodin et al. J. Am. Chem. Soc. 2000, 122, 3932) reflects the pH-dependence of the driving force caused by proton release to the bulk. For the pH-independent ETPT on the other hand, the driving force of the rate-determining ET step is pH-independent and smaller. On the other hand, temperature-dependent data showed that the reorganization energy was higher for CEP, while the pre-exponential factors showed no significant difference between the mechanisms. Thus, the opposing effect of the differences in driving force and reorganization energy determines which of the mechanisms will dominate. Our results show that a concerted mechanism is in general quite likely and provides a low-barrier reaction pathway for weakly exoergonic reactions. In addition, the kinetic isotope effect was much higher for CEP (kH/kD > 10) than for ETPT (kH/kD = 2), consistent with significant changes along the proton reaction coordinate in the rate-determining step of CEP.

Although the mechanism for the transformation of carbon dioxide to formate with copper hydride is well understood, it is not clear how formic acid is ultimately released. Herein, we show how formic acid is formed in the decomposition of the copper formate clusters Cu(II)(HCOO)3 − and Cu(II)2(HCOO)5 −. Infrared irradiation resonant with the antisymmetric C−O stretching mode activates the cluster, resulting in the release of formic acid and carbon dioxide. For the binary cluster, electronic structure calculations indicate that CO2 is eliminated first, through hydride transfer from formate to copper. Formic acid is released via proton‐coupled electron transfer (PCET) to a second formate ligand, evidenced by close to zero partial charge and spin density at the hydrogen atom in the transition state. Concomitantly, the two copper centers are reduced from Cu(II) to Cu(I). Depending on the detailed situation, either PCET or hydrogen atom transfer (HAT) takes place.

Protein Dynamics Coupled to Electron Transfer in Cytochrome C Oxidase from R. Sphaeroides by Time-Resolved Surface-Enhanced 2D-IR-Absorption Spectroscopy

Cytochrome c oxidase (CcO) is the terminal enzyme in the respiratory electron transport chain of aerobic organisms. It catalyses the reduction of atmospheric oxygen to water, and couples this reaction to proton pumping across the membrane; this process generates the electrochemical gradient that subsequently drives the synthesis of ATP. The molecular details of the mechanism by which electron transfer is coupled to proton pumping in CcO is poorly understood. Recent calculations from our group indicate that His291, a ligand of the Cu(B) center of the enzyme, may play the role of the pumping element. In this paper we describe calculations in which a DFT/continuum electrostatic method is used to explore the coupling of the conformational changes of Glu242 residue, the main proton donor of both chemical and pump protons, to its pKa, and the pKa of His291, a putative proton loading site of our pumping model. The computations are done for several redox states of metal centers, different protonation states of Glu242 and His291, and two well-defined conformations of the Glu242 side chain. Thus, in addition to equilibrium redox/protonation states of the catalytic cycle, we also examine the transient and intermediate states. Different dielectric models are employed to investigate the robustness of the results, and their viability in the light of the proposed proton pumping mechanism of CcO. The main results are in agreement with the experimental measurements and support the proposed pumping mechanism. Additionally, the present calculations indicate a possibility of gating through conformational changes of Glu242; namely, in the pumping step, we find that Glu242 needs to be reprotonated before His291 can eject a proton to the P-site of membrane. As a result, the reprotonation of Glu can control proton release from the proton loading site.

Reduction of O₂ by cytochrome c oxidase (COX) is critical to the cellular production of adenosine-5'-triphosphate; COX obtains the four electrons required for this process from ferrocytochrome c. The COX-cytochrome c enzyme-substrate complex is stabilized by electrostatic interactions via carboxylates on COX and lysines on cytochrome c. Conformational changes are believed to play a role in ferrocytochrome c oxidation and release and in rapid intramolecular transfer of electrons within COX, but the details are unclear. To gather specific information about the extent and relevance of conformational changes, we performed bioinformatics studies using the published structures of both proteins. For both proteins, we studied the surface accessibility and energy, as a function of the proteins' oxidation state. The residues of reduced cytochrome c showed greater surface accessibility and were at a higher energy than those of the oxidized cytochrome c. Also, most residues of the core subunits (I, II, and III) of COX showed low accessibility, ∼35%, and compared to the oxidized subunits, the reduced subunits had higher energies. We concluded that substrate binding and dissociation is modulated by specific redox-dependent conformational changes. We further conclude that high energy and structural relaxation of reduced cytochrome c and core COX subunits drive their rapid electron transfer.

Basic concepts and theoretical foundations of broken symmetry (BS) and post BS methods for strongly correlated electron systems (SCES) such as electron-transfer (ET) diradical, multi-center polyradicals with spin frustration are described systematically to elucidate structures, bonding and reactivity of the high-valent transition metal oxo bonds in metalloenzymes: photosystem II (PSII) and cytochrome c oxidase (CcO). BS hybrid DFT (HDFT) and DLPNO coupled-cluster (CC) SD(T0) computations are performed to elucidate electronic and spin states of CaMn4Ox cluster in the key step for oxygen evolution, namely S4 [S3 with Mn(IV) = O + Tyr161-O radical] state of PSII and PM [Fe(IV) = O + HO-Cu(II) + Tyr161-O radical] step for oxygen reduction in CcO. The cycle of water oxidation catalyzed by the CaMn4Ox cluster in PSII and the cycle of oxygen reduction catalyzed by the CuA-Fea-Fea3-CuB cluster in CcO are examined on the theoretical grounds, elucidating similar concerted and/or stepwise proton transfer coupled electron transfer (PT-ET) processes for the four-electron oxidation in PSII and four-electron reduction in CcO. Interplay between theory and experiments have revealed that three electrons in the metal sites and one electron in tyrosine radical site are characteristic for PT-ET in these biological redox reaction systems, indicating no necessity of harmful Mn(V) = O and Fe(V) = O bonds with strong oxyl-radical character. Implications of the computational results are discussed in relation to design of artificial systems consisted of earth abundant transition metals for water oxidation.Graphical abstract