Crystallographic cyanide-probing for cytochrome c oxidase reveals structural bases suggesting that a putative proton transfer H-pathway pumps protons.

Cytochrome c oxidase (CcO) reduces O2 in the O2-reduction site by sequential four-electron donations through the low-potential metal sites (CuA and Fea). Redox-coupled X-ray crystal structural changes have been identified at five distinct sites including Asp51, Arg438, Glu198, the hydroxyfarnesyl ethyl group of heme a, and Ser382, respectively. These sites interact with the putative proton-pumping H-pathway. However, the metal sites responsible for each structural change have not been identified, since these changes were detected as structural differences between the fully reduced and fully oxidized CcOs. Thus, the roles of these structural changes in the CcO function are yet to be revealed. X-ray crystal structures of cyanide-bound CcOs under various oxidation states showed that the O2-reduction site controlled only the Ser382-including site, while the low-potential metal sites induced the other changes. This finding indicates that these low-potential site-inducible structural changes are triggered by sequential electron-extraction from the low-potential sites by the O2-reduction site and that each structural change is insensitive to the oxidation and ligand-binding states of the O2-reduction site. Because the proton/electron coupling efficiency is constant (1:1), regardless of the reaction progress in the O2-reduction site, the structural changes induced by the low-potential sites are assignable to those critically involved in the proton pumping, suggesting that the H-pathway, facilitating these low-potential site-inducible structural changes, pumps protons. Furthermore, a cyanide-bound CcO structure suggests that a hypoxia-inducible activator, Higd1a, activates the O2-reduction site without influencing the electron transfer mechanism through the low-potential sites, kinetically confirming that the low-potential sites facilitate proton pump.

Respiratory complex I in mitochondrial membrane catalyzes oversized ubiquinones.

NADH-ubiquinone (UQ) oxidoreductase (complex I) couples electron transfer from NADH to UQ with proton translocation in its membrane part. The UQ reduction step is key to triggering proton translocation. Structural studies have identified a long, narrow, tunnel-like cavity within complex I, through which UQ may access a deep reaction site. To elucidate the physiological relevance of this UQ-accessing tunnel, we previously investigated whether a series of oversized UQs (OS-UQs), whose tail moiety is too large to enter and transit the narrow tunnel, can be catalytically reduced by complex I using the native enzyme in bovine heart submitochondrial particles (SMPs) and the isolated enzyme reconstituted into liposomes. Nevertheless, the physiological relevance remained unclear because some amphiphilic OS-UQs were reduced in SMPs but not in proteoliposomes, and investigation of extremely hydrophobic OS-UQs was not possible in SMPs. To uniformly assess the electron transfer activities of all OS-UQs with the native complex I, here we present a new assay system using SMPs, which were fused with liposomes incorporating OS-UQ and supplemented with a parasitic quinol oxidase to recycle reduced OS-UQ. In this system, all OS-UQs tested were reduced by the native enzyme, and the reduction was coupled with proton translocation. This finding does not support the canonical tunnel model. We propose that the UQ reaction cavity is flexibly open in the native enzyme to allow OS-UQs to access the reaction site, but their access is obstructed in the isolated enzyme as the cavity is altered by detergent-solubilizing from the mitochondrial membrane.

Calcium-bound structure of bovine cytochrome c oxidase.

The crystal structure of bovine cytochrome c oxidase (CcO) shows a sodium ion (Na+) bound to the surface of subunit I. Changes in the absorption spectrum of heme a caused by calcium ions (Ca2+) are detected as small red shifts, and inhibition of enzymatic activity under low turnover conditions is observed by addition of Ca2+ in a competitive manner with Na+. In this study, we determined the crystal structure of Ca2+-bound bovine CcO in the oxidized and reduced states at 1.7 Å resolution. Although Ca2+ and Na+ bound to the same site of oxidized and reduced CcO, they led to different coordination geometries. Replacement of Na+ with Ca2+ caused a small structural change in the loop segments near the heme a propionate and formyl groups, resulting in spectral changes in heme a. Redox-coupled structural changes observed in the Ca2+-bound form were the same as those previously observed in the Na+-bound form, suggesting that binding of Ca2+ does not severely affect enzymatic function, which depends on these structural changes. The relation between the Ca2+ binding and the inhibitory effect during slow turnover, as well as the possible role of bound Ca2+ are discussed.

Identifying antibiotics based on structural differences in the conserved allostery from mitochondrial heme-copper oxidases.

Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an allosteric inhibitory site buried inside eukaryotic mitochondrial heme-copper oxidases (HCOs), the essential respiratory enzymes for life. The steric conformation around the binding pocket of HCOs is highly conserved among bacteria and eukaryotes, yet the latter has an extra helix. This structural difference in the conserved allostery enabled us to rationally identify bacterial HCO-specific inhibitors: an antibiotic compound against ceftriaxone-resistant Neisseria gonorrhoeae. Molecular dynamics combined with resonance Raman spectroscopy and stopped-flow spectroscopy revealed an allosteric obstruction in the substrate accessing channel as a mechanism of inhibition. Our approach opens fresh avenues in modulating protein functions and broadens our options to overcome AMR.

Effects of Interfacial Interactions on Electrocatalytic Activity of Cytochrome c Oxidase in Biomimetic Lipid Membranes on Gold Electrodes.

Effects of interfacial interactions on the electrocatalytic activity of protein-tethered bilayer lipid membranes (ptBLMs) containing cytochrome c oxidase (CcO) for the oxygen reduction reaction are studied by using protein film electrochemistry and surface-enhanced infrared absorption (SEIRA) spectroscopy. Mammalian CcO was immobilized on a gold electrode via self-assembled monolayers (SAMs) of mixed alkanethiols. The protein orientation on the electrode is controlled by SAM-CcO interactions and is critical to the cytochrome c (cyt c) binding. The CcO-phospholipid and CcO-cyt c interactions modulate the electrocatalytic activity of CcO, and more densely packed ptBLMs show higher electrocatalytic activity. Our study indicates that spectroscopic and electrochemical studies of ptBLMs can provide insights into the effects of relatively weak protein-protein and protein-lipid interactions on the enzymatic activity of transmembrane enzymes.

Diverse reaction behaviors of artificial ubiquinones in mitochondrial respiratory complex I.

The ubiquinone (UQ) reduction step catalyzed by NADH-UQ oxidoreductase (mitochondrial respiratory complex I) is key to triggering proton translocation across the inner mitochondrial membrane. Structural studies have identified a long, narrow, UQ-accessing tunnel within the enzyme. We previously demonstrated that synthetic oversized UQs, which are unlikely to transit this narrow tunnel, are catalytically reduced by native complex I embedded in submitochondrial particles but not by the isolated enzyme. To explain this contradiction, we hypothesized that access of oversized UQs to the reaction site is obstructed in the isolated enzyme because their access route is altered following detergent solubilization from the inner mitochondrial membrane. In the present study, we investigated this using two pairs of photoreactive UQs (pUQm-1/pUQp-1 and pUQm-2/pUQp-2), with each pair having the same chemical properties except for a ∼1.0 Å difference in side-chain widths. Despite this subtle difference, reduction of the wider pUQs by the isolated complex was significantly slower than of the narrower pUQs, but both were similarly reduced by the native enzyme. In addition, photoaffinity-labeling experiments using the four [125I]pUQs demonstrated that their side chains predominantly label the ND1 subunit with both enzymes but at different regions around the tunnel. Finally, we show that the suppressive effects of different types of inhibitors on the labeling significantly changed depending on [125I]pUQs used, indicating that [125I]pUQs and these inhibitors do not necessarily share a common binding cavity. Altogether, we conclude that the reaction behaviors of pUQs cannot be simply explained by the canonical UQ tunnel model.

Theoretical Model of the Far-Red-Light-Adapted Photosystem I Reaction Center of Cyanobacterium Acaryochloris marina Using Chlorophyll d and the Effect of Chlorophyll Exchange.

A theoretical model of the far-red-light-adapted photosystem I (PSI) reaction center (RC) complex of a cyanobacterium, Acaryochloris marina (AmPSI), was constructed based on the exciton theory and the recently identified molecular structure of AmPSI by Hamaguchi et al. (Nat. Commun., 2021, 12, 2333). A. marina performs photosynthesis under the visible to far-red light (400-750 nm), which is absorbed by chlorophyll d (Chl-d). It is in contrast to the situation of all the other oxygenic photosynthetic processes of cyanobacteria and plants, which contains chlorophyll a (Chl-a) that absorbs only 400-700 nm visible light. AmPSI contains 70 Chl-d, 1 Chl-d', 2 pheophytin a (Pheo-a), and 12 carotenoids in the currently available structure. A special pair of Chl-d/Chl-d' acts as the electron donor (P740) and two Pheo-a act as the primary electron acceptor A0 as the counterparts of P700 and Chl-a, respectively, of Chl-a-type PSIs. The exciton Hamiltonian of AmPSI was constructed considering the excitonic coupling strength and site energy shift of individual pigments using the Poisson-TrESP (P-TrESP) and charge density coupling (CDC) methods. The model was constructed to fit the experimentally measured spectra of absorption and circular dichroism (CD) spectra during downhill/uphill excitation energy transfer processes. The constructed theoretical model of AmPSI was further compared with the Chl-a-type PSI of Thermosynechococcus elongatus (TePSI), which contains only Chl-a and Chl-a'. The functional properties of AmPSI and TePSI were further examined by the in silico exchange of Chl-d by Chl-a in the models.

Biochemical and crystallographic studies of monomeric and dimeric bovine cytochrome c oxidase.

Cytochrome c oxidase (CcO), a terminal oxidase in the respiratory chain, catalyzes the reduction of O2 to water coupled with the proton pump across the membrane. Mitochondrial CcO exists in monomeric and dimeric forms, and as a monomer as part of the respiratory supercomplex, although the enzymatic reaction proceeds in the CcO monomer. Recent biochemical and crystallographic studies of monomeric and dimeric CcOs have revealed functional and structural differences among them. In solubilized mitochondrial membrane, the monomeric form is dominant, and a small amount of dimer is observed. The activity of the monomeric CcO is higher than that of the dimer, suggesting that the monomer is the active form. In the structure of monomeric CcO, a hydrogen bond network of water molecules is formed at the entrance of the proton transfer K-pathway, and in dimeric CcO, this network is altered by a cholate molecule binding between monomers. The specific binding of the cholate molecule at the dimer interface suggests that the binding of physiological ligands similar in size or shape to cholate could also trigger dimer formation as a physiological standby form. Because the dimer interface also contains weak interactions of nonspecifically bound lipid molecules, hydrophobic interactions between the transmembrane helices, and a Met-Met interaction between the extramembrane regions, these interactions could support the stabilization of the standby form. Structural analyses also suggest that hydrophobic interactions of cardiolipins bound to the trans-membrane surface of CcO are involved in forming the supercomplex.

Critical roles of the CuB site in efficient proton pumping as revealed by crystal structures of mammalian cytochrome c oxidase catalytic intermediates.

Mammalian cytochrome c oxidase (CcO) reduces O2 to water in a bimetallic site including Fea3 and CuB giving intermediate molecules, termed A-, P-, F-, O-, E-, and R-forms. From the P-form on, each reaction step is driven by single-electron donations from cytochrome c coupled with the pumping of a single proton through the H-pathway, a proton-conducting pathway composed of a hydrogen-bond network and a water channel. The proton-gradient formed is utilized for ATP production by F-ATPase. For elucidation of the proton pumping mechanism, crystal structural determination of these intermediate forms is necessary. Here we report X-ray crystallographic analysis at ∼1.8 Å resolution of fully reduced CcO crystals treated with O2 for three different time periods. Our disentanglement of intermediate forms from crystals that were composed of multiple forms determined that these three crystallographic data sets contained ∼45% of the O-form structure, ∼45% of the E-form structure, and ∼20% of an oxymyoglobin-type structure consistent with the A-form, respectively. The O- and E-forms exhibit an unusually long CuB2+-OH- distance and CuB1+-H2O structure keeping Fea33+-OH- state, respectively, suggesting that the O- and E-forms have high electron affinities that cause the O→E and E→R transitions to be essentially irreversible and thus enable tightly coupled proton pumping. The water channel of the H-pathway is closed in the O- and E-forms and partially open in the R-form. These structures, together with those of the recently reported P- and F-forms, indicate that closure of the H-pathway water channel avoids back-leaking of protons for facilitating the effective proton pumping.

Structure of the far-red light utilizing photosystem I of Acaryochloris marina.

Acaryochloris marina is one of the cyanobacterial species that can use far-red light to drive photochemical reactions for oxygenic photosynthesis. Here, we report the structure of A. marina photosystem I (PSI) reaction center, determined by cryo-electron microscopy at 2.58 Å resolution. The structure reveals an arrangement of electron carriers and light-harvesting pigments distinct from other type I reaction centers. The paired chlorophyll, or special pair (also referred to as P740 in this case), is a dimer of chlorophyll d and its epimer chlorophyll d'. The primary electron acceptor is pheophytin a, a metal-less chlorin. We show the architecture of this PSI reaction center is composed of 11 subunits and we identify key components that help explain how the low energy yield from far-red light is efficiently utilized for driving oxygenic photosynthesis.

The 1.3-Å resolution structure of bovine cytochrome c oxidase suggests a dimerization mechanism.

Cytochrome c oxidase (CcO) in the respiratory chain catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme and proton pumping across the membrane. Although the functional unit of CcO is monomeric, mitochondrial CcO forms a monomer and a dimer, as well as a supercomplex with respiratory complexes I and III. A recent study showed that dimeric CcO has lower activity than monomeric CcO and proposed that dimeric CcO is a standby form for enzymatic activation in the mitochondrial membrane. Other studies have suggested that the dimerization is dependent on specifically arranged lipid molecules, peptide segments, and post-translationally modified amino acid residues. To re-examine the structural basis of dimerization, we improved the resolution of the crystallographic structure to 1.3 Å by optimizing the method for cryoprotectant soaking. The observed electron density map revealed many weakly bound detergent and lipid molecules at the interface of the dimer. The dimer interface also contained hydrogen bonds with tightly bound cholate molecules, hydrophobic interactions between the transmembrane helices, and a Met-Met interaction between the extramembrane regions. These results imply that binding of physiological ligands structurally similar to cholate could trigger dimerization in the mitochondrial membrane and that non-specifically bound lipid molecules at the transmembrane surface between monomers support the stabilization of the dimer. The weak interactions involving the transmembrane helices and extramembrane regions may play a role in positioning each monomer at the correct orientation in the dimer.

Osmotic pressure effects identify dehydration upon cytochrome c-cytochrome c oxidase complex formation contributing to a specific electron pathway formation.

In the electron transfer (ET) reaction from cytochrome c (Cyt c) to cytochrome c oxidase (CcO), we determined the number and sites of the hydration water released from the protein surface upon the formation of the ET complex by evaluating the osmotic pressure dependence of kinetics for the ET from Cyt c to CcO. We identified that ∼20 water molecules were dehydrated in complex formation under turnover conditions, and systematic Cyt c mutations in the interaction site for CcO revealed that nearly half of the released hydration water during the complexation were located around Ile81, one of the hydrophobic amino acid residues near the exposed heme periphery of Cyt c. Such a dehydration dominantly compensates for the entropy decrease due to the association of Cyt c with CcO, resulting in the entropy-driven ET reaction. The energetic analysis of the interprotein interactions in the ET complex predicted by the docking simulation suggested the formation of hydrophobic interaction sites surrounding the exposed heme periphery of Cyt c in the Cyt c-CcO interface (a 'molecular breakwater'). Such sites would contribute to the formation of the hydrophobic ET pathway from Cyt c to CcO by blocking water access from the bulk water phase.

X-ray structures of catalytic intermediates of cytochrome c oxidase provide insights into its O2 activation and unidirectional proton-pump mechanisms.

Cytochrome c oxidase (CcO) reduces O2 to water, coupled with a proton-pumping process. The structure of the O2-reduction site of CcO contains two reducing equivalents, Fe a32+ and CuB1+, and suggests that a peroxide-bound state (Fe a33+-O--O--CuB2+) rather than an O2-bound state (Fe a32+-O2) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fe a32+-O2, whereas Fe a33+-O--O--CuB2+ is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Šresolution. A 1.70 ŠFe-O distance of the ferryl center could best be described as Fe a34+ = O2-, not as Fe a34+-OH- The distance suggests an ∼800-cm-1 Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fe a33+-O--O--CuB2+ state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.

Oversized ubiquinones as molecular probes for structural dynamics of the ubiquinone reaction site in mitochondrial respiratory complex I.

NADH-quinone oxidoreductase (complex I) couples electron transfer from NADH to quinone with proton translocation across the membrane. Quinone reduction is a key step for energy transmission from the site of quinone reduction to the remotely located proton-pumping machinery of the enzyme. Although structural biology studies have proposed the existence of a long and narrow quinone-access channel, the physiological relevance of this channel remains debatable. We investigated here whether complex I in bovine heart submitochondrial particles (SMPs) can catalytically reduce a series of oversized ubiquinones (OS-UQs), which are highly unlikely to transit the narrow channel because their side chain includes a bulky "block" that is ∼13 Šacross. We found that some OS-UQs function as efficient electron acceptors from complex I, accepting electrons with an efficiency comparable with ubiquinone-2. The catalytic reduction and proton translocation coupled with this reduction were completely inhibited by different quinone-site inhibitors, indicating that the reduction of OS-UQs takes place at the physiological reaction site for ubiquinone. Notably, the proton-translocating efficiencies of OS-UQs significantly varied depending on their side-chain structures, suggesting that the reaction characteristics of OS-UQs affect the predicted structural changes of the quinone reaction site required for triggering proton translocation. These results are difficult to reconcile with the current channel model; rather, the access path for ubiquinone may be open to allow OS-UQs to access the reaction site. Nevertheless, contrary to the observations in SMPs, OS-UQs were not catalytically reduced by isolated complex I reconstituted into liposomes. We discuss possible reasons for these contradictory results.

Higd1a improves respiratory function in the models of mitochondrial disorder.

The respiratory chain (RC) transports electrons to form a proton motive force that is required for ATP synthesis in the mitochondria. RC disorders cause mitochondrial diseases that have few effective treatments; therefore, novel therapeutic strategies are critically needed. We previously identified Higd1a as a positive regulator of cytochrome c oxidase (CcO) in the RC. Here, we test that Higd1a has a beneficial effect by increasing CcO activity in the models of mitochondrial dysfunction. We first demonstrated the tissue-protective effects of Higd1a via in situ measurement of mitochondrial ATP concentrations ([ATP]mito) in a zebrafish hypoxia model. Heart-specific Higd1a overexpression mitigated the decline in [ATP]mito under hypoxia and preserved cardiac function in zebrafish. Based on the in vivo results, we examined the effects of exogenous HIGD1A on three cellular models of mitochondrial disease; notably, HIGD1A improved respiratory function that was coupled with increased ATP synthesis and demonstrated cellular protection in all three models. Finally, enzyme kinetic analysis revealed that Higd1a significantly increased the maximal velocity of the reaction between CcO and cytochrome c without changing the affinity between them, indicating that Higd1a is a positive modulator of CcO. These results corroborate that Higd1a, or its mimic, provides therapeutic options for the treatment of mitochondrial diseases.

Purified F-ATP synthase forms a Ca2+-dependent high-conductance channel matching the mitochondrial permeability transition pore.

The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca2+ dissipates the H+ gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca2+ elicits currents matching those of the MMC/PTP. Currents were fully reversible, were stabilized by benzodiazepine 423, a ligand of the OSCP subunit of F-ATP synthase that activates the MMC/PTP, and were inhibited by Mg2+ and adenine nucleotides, which also inhibit the PTP. Channel activity was insensitive to inhibitors of the adenine nucleotide translocase (ANT) and of the voltage-dependent anion channel (VDAC). Native gel-purified oligomers and dimers, but not monomers, gave rise to channel activity. These findings resolve the long-standing mystery of the MMC/PTP and demonstrate that Ca2+ can transform the energy-conserving F-ATP synthase into an energy-dissipating device.

Monomeric structure of an active form of bovine cytochrome c oxidase.

Cytochrome c oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a proton pump across the membrane. In all crystals reported to date, bovine CcO exists as a dimer with the same intermonomer contacts, whereas CcOs and related enzymes from prokaryotes exist as monomers. Recent structural analyses of the mitochondrial respiratory supercomplex revealed that CcO monomer associates with complex I and complex III, indicating that the monomeric state is functionally important. In this study, we prepared monomeric and dimeric bovine CcO, stabilized using amphipol, and showed that the monomer had high activity. In addition, using a newly synthesized detergent, we determined the oxidized and reduced structures of monomer with resolutions of 1.85 and 1.95 Å, respectively. Structural comparison of the monomer and dimer revealed that a hydrogen bond network of water molecules is formed at the entry surface of the proton transfer pathway, termed the K-pathway, in monomeric CcO, whereas this network is altered in dimeric CcO. Based on these results, we propose that the monomer is the activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane. We also determined phospholipid structures based on electron density together with the anomalous scattering effect of phosphorus atoms. Two cardiolipins are found at the interface region of the supercomplex. We discuss formation of the monomeric CcO, dimeric CcO, and supercomplex, as well as their role in regulation of CcO activity.

Low-dose X-ray structure analysis of cytochrome c oxidase utilizing high-energy X-rays.

To investigate the effect of high-energy X-rays on site-specific radiation-damage, low-dose diffraction data were collected from radiation-sensitive crystals of the metal enzyme cytochrome c oxidase. Data were collected at the Structural Biology I beamline (BL41XU) at SPring-8, using 30 keV X-rays and a highly sensitive pixel array detector equipped with a cadmium telluride sensor. The experimental setup of continuous sample translation using multiple crystals allowed the average diffraction weighted dose per data set to be reduced to 58 kGy, and the resulting data revealed a ligand structure featuring an identical bond length to that in the damage-free structure determined using an X-ray free-electron laser. However, precise analysis of the residual density around the ligand structure refined with the synchrotron data showed the possibility of a small level of specific damage, which might have resulted from the accumulated dose of 58 kGy per data set. Further investigation of the photon-energy dependence of specific damage, as assessed by variations in UV-vis absorption spectra, was conducted using an on-line spectrometer at various energies ranging from 10 to 30 keV. No evidence was found for specific radiation damage being energy dependent.

X-ray structural analyses of azide-bound cytochrome c oxidases reveal that the H-pathway is critically important for the proton-pumping activity.

Cytochrome c oxidase (CcO) is the terminal oxidase of cellular respiration, reducing O2 to water and pumping protons. X-ray structural features have suggested that CcO pumps protons via a mechanism involving electrostatic repulsions between pumping protons in the hydrogen-bond network of a proton-conducting pathway (the H-pathway) and net positive charges created upon oxidation of an iron site, heme a (Fe a2+), for reduction of O2 at another iron site, heme a3 (Fe a32+). The protons for pumping are transferred to the hydrogen-bond network from the N-side via the water channel of the H-pathway. Back-leakage of protons to the N-side is thought to be blocked by closure of the water channel. To experimentally test this, we examined X-ray structures of the azide-bound, oxidized bovine CcO and found that an azide derivative (N3--Fe a33+, CuB2+-N3-) induces a translational movement of the heme a3 plane. This was accompanied by opening of the water channel, revealing that Fe a3 and the H-pathway are tightly coupled. The channel opening in the oxidized state is likely to induce back-leakage of pumping protons, which lowers the proton level in the hydrogen-bond network during enzymatic turnover. The proton level decrease weakens the electron affinity of Fe a , if Fe a electrostatically interacts with protons in the hydrogen-bond network. The previously reported azide-induced redox-potential decrease in Fe a supports existence of the electrostatic interaction. In summary, our results indicate that the H-pathway is critical for CcO's proton-pumping function.