Reduction of molecular oxygen in flavodiiron proteins - Catalytic mechanism and comparison to heme-copper oxidases.

The family of flavodiiron proteins (FDPs) plays an important role in the scavenging and detoxification of both molecular oxygen and nitric oxide. Using electrons from a flavin mononucleotide cofactor molecular oxygen is reduced to water and nitric oxide is reduced to nitrous oxide and water. While the mechanism for NO reduction in FDPs has been studied extensively, there is very little information available about O2 reduction. Here we use hybrid density functional theory (DFT) to study the mechanism for O2 reduction in FDPs. An important finding is that a proton coupled reduction is needed after the O2 molecule has bound to the diferrous diiron active site and before the OO bond can be cleaved. This is in contrast to the mechanism for NO reduction, where both NN bond formation and NO bond cleavage occurs from the same starting structure without any further reduction, according to both experimental and computational results. This computational result for the O2 reduction mechanism should be possible to evaluate experimentally. Another difference between the two substrates is that the actual OO bond cleavage barrier is low, and not involved in rate-limiting the reduction process, while the barrier connected with bond cleavage/formation in the NO reduction process is of similar height as the rate-limiting steps. We suggest that these results may be part of the explanation for the generally higher activity for O2 reduction as compared to NO reduction in most FDPs. Comparisons are also made to the O2 reduction reaction in the family of heme­copper oxidases.

The Redox-Active Tyrosine Is Essential for Proton Pumping in Cytochrome c Oxidase.

Cellular respiration involves electron transport via a number of enzyme complexes to the terminal Cytochrome c oxidase (CcO), in which molecular oxygen is reduced to water. The free energy released in the reduction process is used to establish a transmembrane electrochemical gradient, via two processes, both corresponding to charge transport across the membrane in which the enzymes are embedded. First, the reduction chemistry occurring in the active site of CcO is electrogenic, which means that the electrons and protons are delivered from opposite sides of the membrane. Second, the exergonic chemistry is coupled to translocation of protons across the entire membrane, referred to as proton pumping. In the largest subfamily of the CcO enzymes, the A-family, one proton is pumped for every electron needed for the chemistry, making the energy conservation particularly efficient. In the present study, hybrid density functional calculations are performed on a model of the A-family CcOs. The calculations show that the redox-active tyrosine, conserved in all types of CcOs, plays an essential role for the energy conservation. Based on the calculations a reaction mechanism is suggested involving a tyrosyl radical (possibly mixed with tyrosinate character) in all reduction steps. The result is that the free energy released in each reduction step is large enough to allow proton pumping in all reduction steps without prohibitively high barriers when the gradient is present. Furthermore, the unprotonated tyrosine provides a mechanism for coupling the uptake of two protons per electron in every reduction step, i.e. for a secure proton pumping.

The importance of exact exchange-A methodological investigation of NO reduction in heme-copper oxidases.

Significant improvements of the density functional theory (DFT) methodology during the past few decades have made DFT calculations a powerful tool in studies of enzymatic reaction mechanisms. For metalloenzymes, however, there are still concerns about the reliability in the DFT-results. Therefore, a systematic study is performed where the fraction of exact exchange in a hybrid DFT functional is used as a parameter. By varying this parameter, a set of different but related functionals are obtained. The various functionals are applied to one of the reactions occurring in the enzyme family heme-copper oxidases, the reduction of nitric oxide (NO) to nitrous oxide (N2O) and water. The results show that, even though certain parts of the calculated energetics exhibit large variations, the qualitative pictures of the reaction mechanisms are quite stable. Furthermore, it is found that the functional with 15% exact exchange (B3LYP*) gives the best agreement with experimental data for the particular reactions studied. An important aspect of the procedure used is that the computational results are carefully combined with a few more general experimental data to obtain a complete description of the entire catalytic cycle of the reactions studied.

Activation of O2 and NO in heme-copper oxidases - mechanistic insights from computational modelling.

Heme-copper oxidases are transmembrane enzymes involved in aerobic and anaerobic respiration. The largest subgroup contains the cytochrome c oxidases (CcO), which reduce molecular oxygen to water. A significant part of the free energy released in this exergonic process is conserved as an electrochemical gradient across the membrane, via two processes, electrogenic chemistry and proton pumping. A deviant subgroup is the cytochrome c dependent NO reductases (cNOR), which reduce nitric oxide to nitrous oxide and water. This is also an exergonic reaction, but in this case none of the released free energy is conserved. Computational studies applying hybrid density functional theory to cluster models of the bimetallic active sites in the heme-copper oxidases are reviewed. To obtain a reliable description of the reaction mechanisms, energy profiles of the entire catalytic cycles, including the reduction steps have to be constructed. This requires a careful combination of computational results with certain experimental data. Computational studies have elucidated mechanistic details of the chemical parts of the reactions, involving cleavage and formation of covalent bonds, which have not been obtainable from pure experimental investigations. Important insights regarding the mechanisms of energy conservation have also been gained. The computational studies show that the reduction potentials of the active site cofactors in the CcOs are large enough to afford electrogenic chemistry and proton pumping, i.e. efficient energy conservation. These results solve a conflict between different types of experimental data. A mechanism for the proton pumping, involving a specific and crucial role for the active site tyrosine, conserved in all CcOs, is suggested. For the cNORs, the calculations show that the low reduction potentials of the active site cofactors are optimized for fast elimination of the toxic NO molecules. At the same time, the low reduction potentials lead to endergonic reduction steps with high barriers. To prevent even higher barriers, which would lead to a too slow reaction, when the electrochemical gradient across the membrane is present, the chemistry must occur in a non-electrogenic manner. This explains why there is no energy conservation in cNOR.

Role of the Two Metals in the Active Sites of Heme Copper Oxidases-A Study of NO Reduction in cbb3 Cytochrome c Oxidase.

The superfamily of heme copper oxidases reduces molecular oxygen or nitric oxide, and the active sites comprise a high-spin heme group (a3 or b3) and a non-heme metal (CuB or FeB). The cbb3 C family of cytochrome c oxidases, with the high-spin heme b3 and CuB in the active site, is a subfamily of the heme copper oxidases that can reduce both molecular oxygen, which is the main substrate, and nitric oxide. The mechanism for NO reduction in cbb3 oxidase is studied here using hybrid density functional theory and compared to other cytochrome c oxidases (A and B families), with a high-spin heme a3 and CuB in the active site, and to cytochrome c dependent NO reductase, with a high-spin heme b3 and a non-heme FeB in the active site. It is found that the reaction mechanism and the detailed reaction energetics of the cbb3 oxidases are not similar to those of cytochrome c dependent NO reductase, which has the same type of high-spin heme group but a different non-heme metal. This is in contrast to earlier expectations. Instead, the NO reduction mechanism in cbb3 oxidases is very similar to that in the other cytochrome c oxidases, with the same non-heme metal, CuB, and is independent of the type of high-spin heme group. The conclusion is that the type of non-heme metal (CuB or FeB) in the active site of the heme copper oxidases is more important for the reaction mechanisms than the type of high-spin heme, at least for the NO reduction reaction. The reason is that the proton-coupled reduction potentials of the active site cofactors determine the energetics for the NO reduction reaction, and they depend to a larger extent on the non-heme metal. Observed differences in NO reduction reactivity among the various cytochrome c oxidases may be explained by differences outside the BNC, affecting the rate of proton transfer, rather than in the BNC itself.

What can we learn from N2 O isotope data? - Analytics, processes and modelling.

The isotopic composition of nitrous oxide (N2 O) provides useful information for evaluating N2 O sources and budgets. Due to the co-occurrence of multiple N2 O transformation pathways, it is, however, challenging to use isotopic information to quantify the contribution of distinct processes across variable spatiotemporal scales. Here, we present an overview of recent progress in N2 O isotopic studies and provide suggestions for future research, mainly focusing on: analytical techniques; production and consumption processes; and interpretation and modelling approaches. Comparing isotope-ratio mass spectrometry (IRMS) with laser absorption spectroscopy (LAS), we conclude that IRMS is a precise technique for laboratory analysis of N2 O isotopes, while LAS is more suitable for in situ/inline studies and offers advantages for site-specific analyses. When reviewing the link between the N2 O isotopic composition and underlying mechanisms/processes, we find that, at the molecular scale, the specific enzymes and mechanisms involved determine isotopic fractionation effects. In contrast, at plot-to-global scales, mixing of N2 O derived from different processes and their isotopic variability must be considered. We also find that dual isotope plots are effective for semi-quantitative attribution of co-occurring N2 O production and reduction processes. More recently, process-based N2 O isotopic models have been developed for natural abundance and 15 N-tracing studies, and have been shown to be effective, particularly for data with adequate temporal resolution. Despite the significant progress made over the last decade, there is still great need and potential for future work, including development of analytical techniques, reference materials and inter-laboratory comparisons, further exploration of N2 O formation and destruction mechanisms, more observations across scales, and design and validation of interpretation and modelling approaches. Synthesizing all these efforts, we are confident that the N2 O isotope community will continue to advance our understanding of N2 O transformation processes in all spheres of the Earth, and in turn to gain improved constraints on regional and global budgets.

The structure of the oxidized state of cytochrome c oxidase - experiments and theory compared.

Cytochrome c oxidase (CcO), the terminal enzyme in the respiratory chain, reduces molecular oxygen to water. Experimental data on the midpoint potentials of the heme iron/copper active site cofactors do not match the overall reaction energetics, and are also in conflict with the observed efficiency of energy conservation in CcO. Therefore it has been postulated that the ferric/cupric intermediate (the oxidized state) exists in two forms. One form, labelled OH, is presumably involved during catalytic turnover, and should have a high CuB midpoint potential due to a metastable high energy structure. When no more electrons are supplied, the OH state supposedly relaxes to the resting form, labelled O, with a lower energy and a lower midpoint potential. It has been suggested that there is a pure geometrical difference between the OH and O states, obtained by moving a water molecule inside the active site. It is shown here that the difference between the two forms of the oxidized state must be of a more chemical nature. The reason is that all types of geometrically relaxed structures of the oxidized intermediate have similar energies, all with a high proton coupled reduction potential in accordance with the postulated OH state. One hypothesized chemical modification of the OH state is the transfer of an extra proton, possibly internal, into the active site. Such a protonated state has several properties that agree with experimental data on the relaxed oxidized state, including a decreased midpoint potential.

Proton transfer in uncoupled variants of cytochrome c oxidase.

Cytochrome c oxidase is a membrane-bound redox-driven proton pump that harbors two proton-transfer pathways, D and K, which are used at different stages of the reaction cycle. Here, we address the question if a D pathway with a modified energy landscape for proton transfer could take over the role of the K pathway when the latter is blocked by a mutation. Our data indicate that structural alterations near the entrance of the D pathway modulate energy barriers that influence proton transfer to the proton-loading site. The data also suggest that during reduction of the catalytic site, its protonation has to occur via the K pathway and that this proton transfer to the catalytic site cannot take place through the D pathway.

The mechanism for oxygen reduction in the C family cbb3 cytochrome c oxidases - Implications for the proton pumping stoichiometry.

Cytochrome c oxidases (CcOs) couple the exergonic reduction of molecular oxygen to proton pumping across the membrane in which they are embedded, thereby conserving a significant part of the free energy. The A family CcOs are known to pump four protons per oxygen molecule, while there is no consensus regarding the proton pumping stoichiometry for the C family cbb3 oxidases. Hybrid density functional theory is used here to investigate the catalytic mechanism for oxygen reduction in cbb3 oxidases. A surprising result is that the barrier for O O bond cleavage at the mixed valence reduction level seems to be too high compared to the overall reaction rate of the enzyme. It is therefore suggested that the O O bond is cleaved only after the first proton coupled reduction step, and that this reduction step most likely is not coupled to proton pumping. Furthermore, since the cbb3 oxidases have only one proton channel leading to the active site, it is proposed that the activated EH intermediate, suggested to be responsible for proton pumping in one of the reduction steps in the A family, cannot be involved in the catalytic cycle for cbb3, which results in the lack of proton pumping also in the E to R reduction step. In summary, the calculations indicate that only two protons are pumped per oxygen molecule in cbb3 oxidases. However, more experimental information on this divergent enzyme is needed, e.g. whether the flow of electrons resembles that in the other more well-studied CcO families.

Insights into the mechanism of nitric oxide reductase from a FeB -depleted variant.

A key step of denitrification, the reduction of toxic nitric oxide to nitrous oxide, is catalysed by cytochrome c-dependent NO reductase (cNOR). cNOR contains four redox-active cofactors: three hemes and a nonheme iron (FeB ). Heme b3 and FeB constitute the active site, but the specific mechanism of NO-binding events and reduction is under debate. Here, we used a recently constructed, fully folded and hemylated cNOR variant that lacks FeB to investigate the role of FeB during catalysis. We show that in the FeB -less cNOR, binding of both NO and O2 to heme b3 still occurs but further reduction is impaired, although to a lesser degree for O2 than for NO. Implications for the catalytic mechanisms of cNOR are discussed.

Active Site Midpoint Potentials in Different Cytochrome c Oxidase Families: A Computational Comparison.

Cytochrome c oxidase (C cO) is the terminal enzyme in the respiratory electron transport chain, reducing molecular oxygen to water. The binuclear active site in C cO comprises a high-spin heme associated with a CuB complex and a redox active tyrosine. The electron transport in the respiratory chain is driven by increasing midpoint potentials of the involved cofactors, resulting in a release of free energy, which is stored by coupling the electron transfer to proton translocation across a membrane, building up an electrochemical gradient. In this context, the midpoint potentials of the active site cofactors in the C cOs are of special interest, since they determine the driving forces for the individual oxygen reduction steps and thereby affect the efficiency of the proton pumping. It has been difficult to obtain useful information on some of these midpoint potentials from experiments. However, since each of the reduction steps in the catalytic cycle of oxygen reduction to water corresponds to the formation of an O-H bond, they can be calculated with a reasonably high accuracy using quantum chemical methods. From the calculated O-H bond strengths, the proton-coupled midpoint potentials of the active site cofactors can be estimated. Using models representing the different families of C cO's (A, B, and C), the calculations give midpoint potentials that should be relevant during catalytic turnover. The calculations also suggest possible explanations for why some experimentally measured potentials deviate significantly from the calculated ones, i.e., for CuB in all oxidase families, and for heme b3 in the C family.

Mechanisms for enzymatic reduction of nitric oxide to nitrous oxide - A comparison between nitric oxide reductase and cytochrome c oxidase.

Cytochrome c oxidases (CcO) reduce O2 to H2O in the respiratory chain of mitochondria and many aerobic bacteria. In addition, some species of CcO can also reduce NO to N2O and water while others cannot. Here, the mechanism for NO-reduction in CcO is investigated using quantum mechanical calculations. Comparison is made to the corresponding reaction in a "true" cytochrome c-dependent NO reductase (cNOR). The calculations show that in cNOR, where the reduction potentials are low, the toxic NO molecules are rapidly reduced, while the higher reduction potentials in CcO lead to a slower or even impossible reaction, consistent with experimental observations. In both enzymes the reaction is initiated by addition of two NO molecules to the reduced active site, forming a hyponitrite intermediate. In cNOR, N2O can then be formed using only the active-site electrons. In contrast, in CcO, one proton-coupled reduction step most likely has to occur before N2O can be formed, and furthermore, proton transfer is most likely rate-limiting. This can explain why different CcO species with the same heme a3-Cu active site differ with respect to NO reduction efficiency, since they have a varying number and/or properties of proton channels. Finally, the calculations also indicate that a conserved active site valine plays a role in reducing the rate of NO reduction in CcO.

A Systematic DFT Approach for Studying Mechanisms of Redox Active Enzymes.

When DFT has been applied to study mechanisms of redox processes a common procedure has been to study the results for many different functionals. For redox reactions involving the first row transition metals, this approach has given very different results for different functionals. The conclusion has been that DFT cannot be used for these reactions. In the meantime, results with strong predictability have been generated, most noteworthy for photosystem II, where all DFT predictions have been verified by experiments performed later. In order to obtain these predictive results using DFT, an alternative, systematic approach has been used, where the key differences between the results for different functionals can be rationalized by using a single parameter, rather than using the very large number of differences in the functionals.

The mechanism for oxygen reduction in cytochrome c dependent nitric oxide reductase (cNOR) as obtained from a combination of theoretical and experimental results.

Bacterial NO-reductases (NOR) belong to the heme-copper oxidase (HCuO) superfamily, in which most members are O2-reducing, proton-pumping enzymes. This study is one in a series aiming to elucidate the reaction mechanisms of the HCuOs, including the mechanisms for cellular energy conservation. One approach towards this goal is to compare the mechanisms for the different types of HCuOs, cytochrome c oxidase (CcO) and NOR, reducing the two substrates O2 and NO. Specifically in this study, we describe the mechanism for oxygen reduction in cytochrome c dependent NOR (cNOR). Hybrid density functional calculations were performed on large cluster models of the cNOR binuclear active site. Our results are used, together with published experimental information, to construct a free energy profile for the entire catalytic cycle. Although the overall reaction is quite exergonic, we show that during the reduction of molecular oxygen in cNOR, two of the reduction steps are endergonic with high barriers for proton uptake, which is in contrast to oxygen reduction in CcO, where all reduction steps are exergonic. This difference between the two enzymes is suggested to be important for their differing capabilities for energy conservation. An additional result from this study is that at least three of the four reduction steps are initiated by proton transfer to the active site, which is in contrast to CcO, where electrons always arrive before the protons to the active site. The roles of the non-heme metal ion and the redox-active tyrosine in the active site are also discussed.

Splitting of the O-O bond at the heme-copper catalytic site of respiratory oxidases.

Heme-copper oxidases catalyze the four-electron reduction of O2 to H2O at a catalytic site that is composed of a heme group, a copper ion (CuB), and a tyrosine residue. Results from earlier experimental studies have shown that the O-O bond is cleaved simultaneously with electron transfer from a low-spin heme (heme a/b), forming a ferryl state (PR ; Fe4+=O2-, CuB2+-OH-). We show that with the Thermus thermophilus ba3 oxidase, at low temperature (10°C, pH 7), electron transfer from the low-spin heme b to the catalytic site is faster by a factor of ~10 (τ ≅ 11 µs) than the formation of the PR ferryl (τ ≅110 µs), which indicates that O2 is reduced before the splitting of the O-O bond. Application of density functional theory indicates that the electron acceptor at the catalytic site is a high-energy peroxy state [Fe3+-O--O-(H+)], which is formed before the PR ferryl. The rates of heme b oxidation and PR ferryl formation were more similar at pH 10, indicating that the formation of the high-energy peroxy state involves proton transfer within the catalytic site, consistent with theory. The combined experimental and theoretical data suggest a general mechanism for O2 reduction by heme-copper oxidases.

Can Reduction of NO to N2O in Cytochrome c Dependent Nitric Oxide Reductase Proceed through a Trans-Mechanism?

As part of microbial denitrification, NO is reduced to N2O in the membrane bound enzyme nitric oxide reductase, NOR. The N-N coupling occurs in the diiron binuclear active site, BNC, and different mechanisms for this reaction step have been suggested. Computational studies have supported a so-called cis:b3-mechanism, in which the hyponitrite product of the reductive N-N bond formation coordinates with one nitrogen to the heme iron and with both oxygens to the non-heme iron in the BNC. In contrast, experimental results have been interpreted to support a so-called trans-mechanism, in which the hyponitrite intermediate coordinates with one nitrogen atom to each of the two iron ions. Hybrid density functional theory is used here to perform an extensive search for possible intermediates of the NO reduction in the cNOR enzyme. It is found that hyponitrite structures coordinating with their negatively charged oxygens to the positively charged iron ions are the most stable ones. The hyponitrite intermediate involved in the suggested trans-mechanism, which only coordinates with the nitrogens to the iron ions, is found to be prohibitively high in energy, leading to a too slow reaction, which should rule out this mechanism. Furthermore, intermediates binding one NO molecule to each iron ion in the BNC, which have been suggested to initiate the trans-mechanism, are found to be too high in energy to be observable, indicating that the experimentally observed electron paramagnetic resonance signals, taken to support such an iron-nitrosyl dimer intermediate, should be reinterpreted.

Improved free energy profile for reduction of NO in cytochrome c dependent nitric oxide reductase (cNOR).

Quantum chemical calculations play an essential role in the elucidation of reaction mechanisms for redox-active metalloenzymes. For example, the cleavage and the formation of covalent bonds can usually not be described only on the basis of experimental information, but can be followed by the calculations. Conversely, there are properties, like reduction potentials, which cannot be accurately calculated. Therefore, computational and experimental data has to be carefully combined to obtain reliable descriptions of entire catalytic cycles involving electron and proton uptake from donors outside the enzyme. Such a procedure is illustrated here, for the reduction of nitric oxide (NO) to nitrous oxide and water in the membrane enzyme, cytochrome c dependent nitric oxide reductase (cNOR). A surprising experimental observation is that this reaction is nonelectrogenic, which means that no energy is conserved. On the basis of hybrid density functional calculations a free energy profile for the entire catalytic cycle is obtained, which agrees much better with experimental information on the active site reduction potentials than previous ones. Most importantly the energy profile shows that the reduction steps are endergonic and that the entire process is rate-limited by high proton uptake barriers during the reduction steps. This result implies that, if the reaction were electrogenic, it would become too slow when the gradient is present across the membrane. This explains why this enzyme does not conserve any of the free energy released. © 2016 Wiley Periodicals, Inc.

Mechanism of Oxygen Reduction in Cytochrome c Oxidase and the Role of the Active Site Tyrosine.

Cytochrome c oxidase, the terminal enzyme in the respiratory chain, reduces molecular oxygen to water and stores the released energy through electrogenic chemistry and proton pumping across the membrane. Apart from the heme-copper binuclear center, there is a conserved tyrosine residue in the active site (BNC). The tyrosine delivers both an electron and a proton during the O-O bond cleavage step, forming a tyrosyl radical. The catalytic cycle then occurs in four reduction steps, each taking up one proton for the chemistry (water formation) and one proton to be pumped. It is here suggested that in three of the reduction steps the chemical proton enters the center of the BNC, leaving the tyrosine unprotonated with radical character. The reproprotonation of the tyrosine occurs first in the final reduction step before binding the next oxygen molecule. It is also suggested that this reduction mechanism and the presence of the tyrosine are essential for the proton pumping. Density functional theory calculations on large cluster models of the active site show that only the intermediates with the proton in the center of the BNC and with an unprotonated tyrosyl radical have a high electron affinity of similar size as the electron donor, which is essential for the ability to take up two protons per electron and thus for the proton pumping. This type of reduction mechanism is also the only one that gives a free energy profile in accordance with experimental observations for the amount of proton pumping in the working enzyme.

Protonation of the binuclear active site in cytochrome c oxidase decreases the reduction potential of CuB.

One of the remaining mysteries regarding the respiratory enzyme cytochrome c oxidase is how proton pumping can occur in all reduction steps in spite of the low reduction potentials observed in equilibrium titration experiments for two of the active site cofactors, CuB(II) and Fea3(III). It has been speculated that, at least the copper cofactor can acquire two different states, one metastable activated state occurring during enzyme turnover, and one relaxed state with lower energy, reached only when the supply of electrons stops. The activated state should have a transiently increased CuB(II) reduction potential, allowing proton pumping. The relaxed state should have a lower reduction potential, as measured in the titration experiments. However, the structures of these two states are not known. Quantum mechanical calculations show that the proton coupled reduction potential for CuB is inherently high in the active site as it appears after reaction with oxygen, which explains the observed proton pumping. It is suggested here that, when the flow of electrons ceases, a relaxed resting state is formed by the uptake of one extra proton, on top of the charge compensating protons delivered in each reduction step. The extra proton in the active site decreases the proton coupled reduction potential for CuB by almost half a volt, leading to agreement with titration experiments. Furthermore, the structure for the resting state with an extra proton is found to have a hydroxo-bridge between CuB(II) and Fea3(III), yielding a magnetic coupling that can explain the experimentally observed EPR silence.

How cytochrome c oxidase can pump four protons per oxygen molecule at high electrochemical gradient.

Experiments have shown that the A-family cytochrome c oxidases pump four protons per oxygen molecule, also at a high electrochemical gradient. This has been considered a puzzle, since two of the reduction potentials involved, Cu(II) and Fe(III), were estimated from experiments to be too low to afford proton pumping at a high gradient. The present quantum mechanical study (using hybrid density functional theory) suggests a solution to this puzzle. First, the calculations show that the charge compensated Cu(II) potential for CuB is actually much higher than estimated from experiment, of the same order as the reduction potentials for the tyrosyl radical and the ferryl group, which are also involved in the catalytic cycle. The reason for the discrepancy between theory and experiment is the very large uncertainty in the experimental observations used to estimate the equilibrium potentials, mainly caused by the lack of methods for direct determination of reduced CuB. Second, the calculations show that a high energy metastable state, labeled EH, is involved during catalytic turnover. The EH state mixes the low reduction potential of Fe(III) in heme a3 with another, higher potential, here suggested to be that of the tyrosyl radical, resulting in enough exergonicity to allow proton pumping at a high gradient. In contrast, the corresponding metastable oxidized state, OH, is not significantly higher in energy than the resting state, O. Finally, to secure the involvement of the high energy EH state it is suggested that only one proton is taken up via the K-channel during catalytic turnover.