Dynamic water networks in cytochrome cbb(3) oxidase

Heme-copper oxidases (HCOs) are terminal electron acceptors in aerobic respiration. They catalyze the reduction of molecular oxygen to water with concurrent pumping of protons across the mitochondrial and bacterial membranes. Protons required for oxygen reduction chemistry and pumping are transferred through proton uptake channels. Recently, the crystal structure of the first C-type member of the HCO superfamily was resolved [Buschmann et al. Science 329 (2010) 327-330], but crystallographic water molecules could not be identified. Here we have used molecular dynamics (MD) simulations, continuum electrostatic approaches, and quantum chemical cluster calculations to identify proton transfer pathways in cytochrome cbb(3). In MD simulations we observe formation of stable water chains that connect the highly conserved Glu323 residue on the proximal side of heme b(3) both with the N- and the P-sides of the membrane. We propose that such pathways could be utilized for redox-coupled proton pumping in the C-type oxidases. Electrostatics and quantum chemical calculations suggest an increased proton affinity of Glu323 upon reduction of high-spin heme b(3). Protonation of Glu323 provides a mechanism to tune the redox potential of heme b(3) with possible implications for proton pumping.     

Redox-coupled proton pumping in cytochrome c oxidase: Further insights from computer simulation

The membrane-bound enzyme cytochrome c oxidase, the terminal member in the respiratory chain, converts oxygen into water and generates an electrochemical gradient by coupling the electron transfer to proton pumping across the membrane. Here we have investigated the dynamics of an excess proton and the surrounding protein environment near the active sites. The multi-state empirical valence bond (MS-EVB) molecular dynamics method was used to simulate the explicit dynamics of proton transfer through the critically important hydrophobic channel between Glu242 (bovine notation) and the D-propionate of heme a₃ (PRDa₃) for the first time. The results from these molecular dynamics simulations indicate that the PRDa₃ can indeed re-orientate and dissociate from Arg438, despite the high stability of such an ion pair, and has the ability to accept protons via bound water molecules. Any large conformational change of the adjacent heme a D-propionate group is, however, sterically blocked directly by the protein. Free energy calculations of the PRDa₃ side chain isomerization and the proton translocation between Glu242 and the PRDa₃ site have also been performed. The results exhibit a redox state-dependent dynamical behavior and indicate that reduction of the low-spin heme a may initiate internal transfer of the pumped proton from Glu242 to the PRDa₃ site.     

Redox-coupled proton transfer in the active site of cytochrome cbb3

Cytochrome cbb₃ is a distinct member of the superfamily of respiratory heme-copper oxidases, and is responsible for driving the respiratory chain in many pathogenic bacteria. Like the canonical heme-copper oxidases, cytochrome cbb₃ reduces oxygen to water and couples the released energy to pump protons across the bacterial membrane. Homology modeling and recent electron paramagnetic resonance (EPR) studies on wild type and a mutant cbb₃ enzyme [V. Rauhamäki et al. J. Biol. Chem. 284 (2009) 11301-11308] have led us to perform high-level quantum chemical calculations on the active site. These calculations bring molecular insight into the unique hydrogen bonding between the proximal histidine ligand of heme b₃ and a conserved glutamate, and indicate that the catalytic mechanism involves redox-coupled proton transfer between these residues. The calculated spin densities give insight in the difference in EPR spectra for the wild type and a recently studied E383Q-mutant cbb₃-enzyme. Furthermore, we show that the redox-coupled proton movement in the proximal cavity of cbb₃-enzymes contributes to the low redox potential of heme b₃, and suggest its potential implications for the high apparent oxygen affinity of these enzymes.     

Substrate binding and the catalytic reactions in cbb3-type oxidases: The lipid membrane modulates ligand binding

Heme–copper oxidases (HCuOs) are the terminal components of the respiratory chain in the mitochondrial membrane or the cell membrane in many bacteria. These enzymes reduce oxygen to water and use the free energy from this reaction to maintain a proton-motive force across the membrane in which they are embedded. The heme–copper oxidases of the cbb₃-type are only found in bacteria, often pathogenic ones since they have a low K_m for O₂, enabling the bacteria to colonize semi-anoxic environments. Cbb₃-type (C) oxidases are highly divergent from the mitochondrial-like aa₃-type (A) oxidases, and within the heme–copper oxidase family, cbb₃ is the closest relative to the most divergent member, the bacterial nitric oxide reductase (NOR). Nitric oxide reductases reduce NO to N₂O without coupling the reaction to the generation of any electrochemical proton gradient. The significant structural differences between A- and C-type heme–copper oxidases are manifested in the lack in cbb₃ of most of the amino acids found to be important for proton pumping in the A-type, as well as in the different binding characteristics of ligands such as CO, O₂ and NO. Investigations of the reasons for these differences at a molecular level have provided insights into the mechanism of O₂ and NO reduction as well as the proton-pumping mechanism in all heme–copper oxidases. In this paper, we discuss results from these studies with the focus on the relationship between proton transfer and ligand binding and reduction. In addition, we present new data, which show that CO binding to one of the c-type hemes of CcoP is modulated by protein–lipid interactions in the membrane. These results show that the heme c-CO binding can be used as a probe of protein–membrane interactions in cbb₃ oxidases, and possible physiological consequences for this behavior are discussed.     

Variable proton-pumping stoichiometry in structural variants of cytochrome c oxidase

Cytochrome c oxidase is a multisubunit membrane-bound enzyme, which catalyzes oxidation of four molecules of cytochrome c²⁺ and reduction of molecular oxygen to water. The electrons are taken from one side of the membrane while the protons are taken from the other side. This topographical arrangement results in a charge separation that is equivalent to moving one positive charge across the membrane for each electron transferred to O₂. In this reaction part of the free energy available from O₂ reduction is conserved in the form of an electrochemical proton gradient. In addition, part of the free energy is used to pump on average one proton across the membrane per electron transferred to O₂. Our understanding of the molecular design of the machinery that couples O₂ reduction to proton pumping in oxidases has greatly benefited from studies of so called “uncoupled” structural variants of the oxidases. In these uncoupled oxidases the catalytic O₂-reduction reaction may display the same rates as in the wild-type CytcO, yet the electron/proton transfer to O₂ is not linked to proton pumping. One striking feature of all uncoupled variants studied to date is that the (apparent) pK_a of a Glu residue, located deeply within a proton pathway, is either increased or decreased (from 9.4 in the wild-type oxidase). The altered pK_a presumably reflects changes in the local structural environment of the residue and because the Glu residue is found near the catalytic site as well as near a putative exit pathway for pumped protons these changes are presumably important for controlling the rates and trajectories of the proton transfer. In this paper we summarize data obtained from studies of uncoupled structural oxidase variants and present a hypothesis that in quantitative terms offers a link between structural changes, modulation of the apparent pK_a and uncoupling of proton pumping from O₂ reduction.     

Proton transfer in ba3 cytochrome c oxidase from Thermus thermophilus

The respiratory heme-copper oxidases catalyze reduction of O₂ to H₂O, linking this process to transmembrane proton pumping. These oxidases have been classified according to the architecture, location and number of proton pathways. Most structural and functional studies to date have been performed on the A-class oxidases, which includes those that are found in the inner mitochondrial membrane and bacteria such as Rhodobacter sphaeroides and Paracoccus denitrificans (aa₃-type oxidases in these bacteria). These oxidases pump protons with a stoichiometry of one proton per electron transferred to the catalytic site. The bacterial A-class oxidases use two proton pathways (denoted by letters D and K, respectively), for the transfer of protons to the catalytic site, and protons that are pumped across the membrane. The B-type oxidases such as, for example, the ba₃ oxidase from Thermus thermophilus, pump protons with a lower stoichiometry of 0.5 H⁺/electron and use only one proton pathway for the transfer of all protons. This pathway overlaps in space with the K pathway in the A class oxidases without showing any sequence homology though. Here, we review the functional properties of the A- and the B-class ba₃ oxidases with a focus on mechanisms of proton transfer and pumping. This article is part of a Special Issue entitled: Respiratory Oxidases.     

The origin of the FeIV = O intermediates in cytochrome aa3 oxidase

The dioxygen reduction mechanism in cytochrome oxidases relies on proton control of the electron transfer events that drive the process. Proton delivery and proton channels in the protein that are relevant to substrate reduction and proton pumping are considered, and the current status of this area is summarized. We propose a mechanism in which the coupling of the oxygen reduction chemistry to proton translocation (P → F transition) is related to the properties of two groups of highly conserved residues, namely, His411/G386-T389 and the heme a₃–propionateA–D399–H403 chain. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Allosteric interactions and proton conducting pathways in proton pumping aa3 oxidases: Heme a as a key coupling element

In this paper allosteric interactions in protonmotive heme aa₃ terminal oxidases of the respiratory chain are dealt with. The different lines of evidence supporting the key role of H⁺/e^? coupling (redox Bohr effect) at the low spin heme a in the proton pump of the bovine oxidase are summarized. Results are presented showing that the I-R54M mutation in P. denitrificans aa₃ oxidase, which decreases by more than 200 mV the E_m of heme a, inhibits proton pumping. Mutational aminoacid replacement in proton channels, at the negative (N) side of membrane-inserted prokaryotic aa₃ oxidases, as well as Zn^2 + binding at this site in the bovine oxidase, uncouples proton pumping. This effect appears to result from alteration of the structural/functional device, closer to the positive, opposite (P) surface, which separates pumped protons from those consumed in the reduction of O₂ to 2 H₂O. This article is part of a Special Issue entitled: Respiratory Oxidases.     

The H channel is not a proton transfer path in yeast cytochrome c oxidase

Cytochrome c oxidases (CcOs) in the respiratory chains of mitochondria and bacteria are primary consumers of molecular oxygen, converting it to water with the concomitant pumping of protons across the membrane to establish a proton electrochemical gradient. Despite a relatively well understood proton pumping mechanism of bacterial CcOs, the role of the H channel in mitochondrial forms of CcO remains debated. Here, we used site-directed mutagenesis to modify a central residue of the lower span of the H channel, Q413, in the genetically tractable yeast Saccharomyces cerevisiae. Exchange of Q413 to several different amino acids showed no effect on rates and efficiencies of respiratory cell growth, and redox potential measurements indicated minimal electrostatic interaction between the 413 locus and the nearest redox active component heme a. These findings clearly exclude a primary role of this section of the H channel in proton pumping in yeast CcO. In agreement with the experimental data, atomistic molecular dynamics simulations and continuum electrostatic calculations on wildtype and mutant yeast CcOs highlight potential bottlenecks in proton transfer through this route. Our data highlight the preference for neutral residues in the 413 locus, precluding sufficient hydration for formation of a proton conducting wire.     

Transmembrane proton translocation by cytochrome c oxidase

Respiratory heme-copper oxidases are integral membrane proteins that catalyze the reduction of molecular oxygen to water using electrons donated by either quinol (quinol oxidases) or cytochrome c (cytochrome c oxidases, CcOs). Even though the X-ray crystal structures of several heme-copper oxidases and results from functional studies have provided significant insights into the mechanisms of O₂-reduction and, electron and proton transfer, the design of the proton-pumping machinery is not known. Here, we summarize the current knowledge on the identity of the structural elements involved in proton transfer in CcO. Furthermore, we discuss the order and timing of electron-transfer reactions in CcO during O₂ reduction and how these reactions might be energetically coupled to proton pumping across the membrane.     

Hydrogen-Bonded Networks Along and Bifurcation of the E-Pathway in Quinol:Fumarate Reductase

The E-pathway of transmembrane proton transfer has been demonstrated previously to be essential for catalysis by the diheme-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes. Two constituents of this pathway, Glu-C180 and heme b_D ring C (b_D-C-) propionate, have been validated experimentally. Here, we identify further constituents of the E-pathway by analysis of molecular dynamics simulations. The redox state of heme groups has a crucial effect on the connectivity patterns of mobile internal water molecules that can transiently support proton transfer from the b_D-C-propionate to Glu-C180. The short H-bonding paths formed in the reduced states can lead to high proton conduction rates and thus provide a plausible explanation for the required opening of the E-pathway in reduced QFR. We found evidence that the b_D-C-propionate group is the previously postulated branching point connecting proton transfer to the E-pathway from the quinol-oxidation site via interactions with the heme b_D ligand His-C44. An essential functional role of His-C44 is supported experimentally by site-directed mutagenesis resulting in its replacement with Glu. Although the H44E variant enzyme retains both heme groups, it is unable to catalyze quinol oxidation. All results obtained are relevant to the QFR enzymes from the human pathogens Campylobacter jejuni and Helicobacter pylori.     

Redox-coupled proton pumping in cytochrome c oxidase: further insights from computer simulation

The membrane-bound enzyme cytochrome c oxidase, the terminal member in the respiratory chain, converts oxygen into water and generates an electrochemical gradient by coupling the electron transfer to proton pumping across the membrane. Here we have investigated the dynamics of an excess proton and the surrounding protein environment near the active sites. The multi-state empirical valence bond (MS-EVB) molecular dynamics method was used to simulate the explicit dynamics of proton transfer through the critically important hydrophobic channel between Glu242 (bovine notation) and the D-propionate of heme a3 (PRDa3) for the first time. The results from these molecular dynamics simulations indicate that the PRDa3 can indeed re-orientate and dissociate from Arg438, despite the high stability of such an ion pair, and has the ability to accept protons via bound water molecules. Any large conformational change of the adjacent heme a D-propionate group is, however, sterically blocked directly by the protein. Free energy calculations of the PRDa3 side chain isomerization and the proton translocation between Glu242 and the PRDa3 site have also been performed. The results exhibit a redox state-dependent dynamical behavior and indicate that reduction of the low-spin heme a may initiate internal transfer of the pumped proton from Glu242 to the PRDa3 site.     

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

Cytochrome c Oxidase (CcO) is the terminal electron acceptor in aerobic respiratory chain, reducing O₂ to water. The released free energy is stored by pumping protons through the protein, maintaining the transmembrane electrochemical gradient. Protons are held transiently in a proton loading site (PLS) that binds and releases protons driven by the electron transfer reaction cycle. Multi-Conformation Continuum Electrostatics (MCCE) was applied to crystal structures and Molecular Dynamics snapshots of the B-type Thermus thermophilus CcO. Six residues are identified as the PLS, binding and releasing protons as the charges on heme b and the binuclear center are changed: the heme a₃ propionic acids, Asp287, Asp372, His376 and Glu126B. The unloaded state has one proton and the loaded state two protons on these six residues. Different input structures, modifying the PLS conformation, show different proton distributions and result in different proton pumping behaviors. One loaded and one unloaded protonation states have the loaded/unloaded states close in energy so the PLS binds and releases a proton through the reaction cycle. The alternative proton distributions have state energies too far apart to be shifted by the electron transfers so are locked in loaded or unloaded states. Here the protein can use active states to load and unload protons, but has nearby trapped states, which stabilize PLS protonation state, providing new ideas about the CcO proton pumping mechanism. The distance between the PLS residues Asp287 and His376 correlates with the energy difference between loaded and unloaded states.     

The Protein Effect in the Structure of Two Ferryl-Oxo Intermediates at the Same Oxidation Level in the Heme Copper Binuclear Center of Cytochrome c Oxidase

Identification of the intermediates and determination of their structures in the reduction of dioxygen to water by cytochrome c oxidase (CcO) are particularly important to understanding both O₂ activation and proton pumping by the enzyme. In this work, we report the products of the rapid reaction of O₂ with the mixed valence form (Cu_A²⁺, heme a³⁺, heme a₃²⁺-Cu_B¹⁺) of the enzyme. The resonance Raman results show the formation of two ferryl-oxo species with characteristic Fe(IV)=O stretching modes at 790 and 804 cm⁻¹ at the peroxy oxidation level (P_M). Density functional theory calculations show that the protein environment of the proximal H-bonded His-411 determines the strength of the distal Fe(IV)=O bond. In contrast to previous proposals, the P_M intermediate is also formed in the reaction of Y167F with O₂. These results suggest that in the fully reduced enzyme, the proton pumping ν_Fe(IV)=O = 804 cm⁻¹ to ν_Fe(IV)=O = 790 cm⁻¹ transition (P→F, where P is peroxy and F is ferryl) is triggered not only by electron transfer from heme a to heme a₃ but also by the formation of the H-bonded form of the His-411-Fe(IV)=O conformer in the proximal site of heme a₃. The implications of these results with respect to the role of an O=Fe(IV)-His-411-H-bonded form to the ring A propionate of heme a₃-Asp-399-H₂O site and, thus, to the exit/output proton channel (H₂O) pool during the proton pumping P→F transition are discussed. We propose that the environment proximal to the heme a₃ controls the spectroscopic properties of the ferryl intermediates in cytochrome oxidases.     

A structural and functional perspective on the evolution of the heme–copper oxidases

The heme–copper oxidases (HCOs) catalyze the reduction of O₂ to water, and couple the free energy to proton pumping across the membrane. HCOs are divided into three sub-classes, A, B and C, whose order of emergence in evolution has been controversial. Here we have analyzed recent structural and functional data on HCOs and their homologues, the nitric oxide reductases (NORs). We suggest that the C-type oxidases are ancient enzymes that emerged from the NORs. In contrast, the A-type oxidases are the most advanced from both structural and functional viewpoints, which we interpret as evidence for having evolved later.     

Spectroscopic study on the communication between a heme a3 propionate, Asp399 and the binuclear center of cytochrome c oxidase from Paracoccus denitrificans

The proton pumping mechanism of cytochrome c oxidase on a molecular level is highly disputed. Recently theoretical calculations and real time electron transfer measurements indicated the involvement of residues in the vicinity of the ring A propionate of heme a₃, including Asp399 and the Cu_B ligands His 325, 326. In this study we probed the interaction of Asp399 with the binuclear center and characterize the protonation state of its side chain. Redox induced FTIR difference spectra of mutations at the site in direct comparison to wild type, indicate that below pH 5 Asp 399 displays signals typical for the deprotonation of the acidic residue with reduction of the enzyme. Interestingly at a pH higher than 5, no contributions from Asp 399 are evident. In order to probe the interaction of the site with the binuclear center we followed the rebinding of CO by infrared spectroscopy for mutations on residue Asp399 to Glu, Asn and Leu. Previously different CO conformers have been identified for bacterial cytochrome c oxidases, and its pH dependent behaviour discussed to be relevant for catalysis. Interestingly we observe the lack of this pH dependency and a strong influence on the observable conformers for all mutants studied here, clearly suggesting a communication of the site with the heme-copper center and the nearby histidine residues.     

Towards the mechanism of proton pumping by the haem-copper oxidases

The haem-copper oxidases comprise a large family of enzymes that is widespread among aerobic organisms. These remarkable membrane-bound proteins catalyse the respiratory reduction of dioxygen to water, and conserve free energy from this reaction by operating as proton pumps. The mechanism of redox-dependent proton translocation has been elusive despite the availability of high resolution crystal structures from several oxidases. Here, we discuss some recent as well as some older results that may shed light on this mechanism. We conclude that proton-pumping is initiated by vectorial proton transfer from a conserved glutamic acid (Glu242 in the bovine enzyme) to a proton acceptor above the haem groups, and that this primary event is mechanistically coupled to electron transfer from haem a to the binuclear haem a₃/Cu_B centre. Subsequently, Glu242 is reprotonated from the negatively charged side of the membrane. Next this proton is transferred to the binuclear site to complete the chemistry, Glu242 is reprotonated once more, and the “prepumped” proton is ejected on the opposite side of the membrane. The different kinetics of electron-coupled proton transfer in different steps of the catalytic cycle may be related to differences in the driving force due to different E_m values of the electron acceptor in the binuclear site.     

Energy diagrams and mechanism for proton pumping in cytochrome c oxidase

The powerful technique of energy diagrams has been used to analyze the mechanism for proton pumping in cytochrome c oxidase. Energy levels and barriers are derived starting out from recent kinetic experiments for the O to E transition, and are then refined using general criteria and a few additional experimental facts. Both allowed and non-allowed pathways are obtained in this way. A useful requirement is that the forward and backward rate should approach each other for the full membrane gradient. A key finding is that an electron on heme a (or the binuclear center) must have a significant lowering effect on the barrier for proton uptake, in order to prevent backflow from the pump-site to the N-side. While there is no structural gating in the present mechanism, there is thus an electronic gating provided by the electron on heme a. A quantitative analysis of the energy levels in the diagrams, leads to Prop-A of heme a₃ as the most likely position for the pump-site, and the Glu278 region as the place for the transition state for proton uptake. Variations of key redox potentials and pK_a values during the pumping process are derived for comparison to experiments.     

Concerted involvement of cooperative proton–electron linkage and water production in the proton pump of cytochrome c oxidase

In cytochrome c oxidase, oxido-reductions of heme a/Cu_A and heme a₃/Cu_B are cooperatively linked to proton transfer at acid/base groups in the enzyme. H⁺/e⁻ cooperative linkage at Fe_a3/Cu_B is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H⁺/e⁻ linkage at heme a (and Cu_A). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H₂O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/Cu_A and heme a₃/Cu_B in the soluble oxidase; (iii) H⁺ release in the external phase (i.e. H⁺ pumping) associated with the oxidative (R → O transition), reductive (O → R transition) and a full catalytic cycle (R → O → R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H⁺/e⁻ linkage at heme a/Cu_A and heme a₃/Cu_B with acid/base clusters, C₁ and C₂ respectively, and protonmotive steps of the reduction of O₂ to water are involved in proton pumping.     

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.