Metformin, phenformin, and galegine inhibit complex IV activity and reduce glycerol-derived gluconeogenesis.

SignificanceMetformin is the most commonly prescribed drug for the treatment of type 2 diabetes mellitus, yet the mechanism by which it lowers plasma glucose concentrations has remained elusive. Most studies to date have attributed metformin's glucose-lowering effects to inhibition of complex I activity. Contrary to this hypothesis, we show that inhibition of complex I activity in vitro and in vivo does not reduce plasma glucose concentrations or inhibit hepatic gluconeogenesis. We go on to show that metformin, and the related guanides/biguanides, phenformin and galegine, inhibit complex IV activity at clinically relevant concentrations, which, in turn, results in inhibition of glycerol-3-phosphate dehydrogenase activity, increased cytosolic redox, and selective inhibition of glycerol-derived hepatic gluconeogenesis both in vitro and in vivo.

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.

Production of recombinant His-tagged triple-FLAG peptide in Brevibacillus choshinensis and its utilization as an easy-to-remove affinity peptide.

Triple-FLAG (3 × FLAG)-tagged proteins can be affinity purified through binding to an anti-FLAG antibody and competitive elution with excess free 3 × FLAG peptide. To expand the availability of the 3 × FLAG purification system, we produced a recombinant His-tagged 3 × FLAG peptide in Brevibacillus choshinensis. The screening of connecting linkers between His-tag and the 3 × FLAG peptide, culture containers, and culture media showed that the His-tagged 3 × FLAG peptide with an LA linker was most expressed in 2SY medium using a baffled shake flask. The peptide was affinity-purified to give a yield of about 25 mg/L of culture. The peptide was effective for eluting 3 × FLAG-tagged α-amylase from anti-FLAG magnetic beads. Finally, the peptide remaining in the amylase fraction was removed by His-tag affinity purification. These results show that the recombinant His-tagged 3 × FLAG peptide can function as an easy-to-remove affinity peptide in the 3 × FLAG purification system.

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.

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.

Complex structure of cytochrome c-cytochrome c oxidase reveals a novel protein-protein interaction mode.

Mitochondrial cytochrome c oxidase (CcO) transfers electrons from cytochrome c (Cyt.c) to O2 to generate H2O, a process coupled to proton pumping. To elucidate the mechanism of electron transfer, we determined the structure of the mammalian Cyt.c-CcO complex at 2.0-Å resolution and identified an electron transfer pathway from Cyt.c to CcO. The specific interaction between Cyt.c and CcO is stabilized by a few electrostatic interactions between side chains within a small contact surface area. Between the two proteins are three water layers with a long inter-molecular span, one of which lies between the other two layers without significant direct interaction with either protein. Cyt.c undergoes large structural fluctuations, using the interacting regions with CcO as a fulcrum. These features of the protein-protein interaction at the docking interface represent the first known example of a new class of protein-protein interaction, which we term "soft and specific". This interaction is likely to contribute to the rapid association/dissociation of the Cyt.c-CcO complex, which facilitates the sequential supply of four electrons for the O2 reduction reaction.

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.

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.

The Mg2+-containing Water Cluster of Mammalian Cytochrome c Oxidase Collects Four Pumping Proton Equivalents in Each Catalytic Cycle.

Bovine heart cytochrome c oxidase (CcO) pumps four proton equivalents per catalytic cycle through the H-pathway, a proton-conducting pathway, which includes a hydrogen bond network and a water channel operating in tandem. Protons are transferred by H3O+ through the water channel from the N-side into the hydrogen bond network, where they are pumped to the P-side by electrostatic repulsion between protons and net positive charges created at heme a as a result of electron donation to O2 bound to heme a3 To block backward proton movement, the water channel remains closed after O2 binding until the sequential four-proton pumping process is complete. Thus, the hydrogen bond network must collect four proton equivalents before O2 binding. However, a region with the capacity to accept four proton equivalents was not discernable in the x-ray structures of the hydrogen bond network. The present x-ray structures of oxidized/reduced bovine CcO are improved from 1.8/1.9 to 1.5/1.6 Å resolution, increasing the structural information by 1.7/1.6 times and revealing that a large water cluster, which includes a Mg2+ ion, is linked to the H-pathway. The cluster contains enough proton acceptor groups to retain four proton equivalents. The redox-coupled x-ray structural changes in Glu198, which bridges the Mg2+ and CuA (the initial electron acceptor from cytochrome c) sites, suggest that the CuA-Glu198-Mg2+ system drives redox-coupled transfer of protons pooled in the water cluster to the H-pathway. Thus, these x-ray structures indicate that the Mg2+-containing water cluster is the crucial structural element providing the effective proton pumping in bovine CcO.

Roles of Mn-catalase and a possible heme peroxidase homologue in protection from oxidative stress in Thermus thermophilus.

Hydrogen peroxide (H2O2) produces hydroxyl radicals that directly attack a variety of biomolecules and cause severe cellular dysfunction. An extremely thermophilic bacterium, Thermus thermophilus HB8, possesses at least three enzymes that can scavenge H2O2: manganese-containing catalase (TTHA0122, MnCAT), a possible peroxiredoxin homologue (TTHA1300), and a possible heme peroxidase (HPX) homologue (TTHA1714). To investigate the roles of these proteins, we attempted to disrupt each of these genes in T. thermophilus HB8. Although we were able to completely disrupt ttha1300, we were unable to completely delete ttha0122 and ttha1714 because of polyploidy. Quantitative real-time PCR showed that, compared to the wild type, 31 % of ttha0122 and 11 % of ttha1714 remained in the ∆ttha0122 and ∆ttha1714 disruption mutants, respectively. Mutants with reduced levels of ttha0122 or ttha1714 exhibited a significant increase in spontaneous mutation frequency. ∆ttha1714 grew slower than the wild type under normal conditions. ∆ttha0122 grew very poorly after exposure to H2O2. Moreover, ∆ttha0122 did not show H2O2-scavenging activity, whereas ∆ttha1300 and ∆ttha1714 scavenged H2O2, a property similar to that exhibited by the wild type. MnCAT purified from T. thermophilus HB8 cells scavenged H2O2 in vitro. The recombinant form of the possible HPX homologue, reconstituted with hemin, showed peroxidase activity with H2O2 as an oxidant substrate. Based on these results, we propose that not only MnCAT but also the possible HPX homologue is involved in protecting the cell from oxidative stress in T. thermophilus.

Thermostable Mismatch-Recognizing Protein MutS Suppresses Nonspecific Amplification during Polymerase Chain Reaction (PCR).

Polymerase chain reaction (PCR)-related technologies are hampered mainly by two types of error: nonspecific amplification and DNA polymerase-generated mutations. Here, we report that both errors can be suppressed by the addition of a DNA mismatch-recognizing protein, MutS, from a thermophilic bacterium. Although it had been expected that MutS has a potential to suppress polymerase-generated mutations, we unexpectedly found that it also reduced nonspecific amplification. On the basis of this finding, we propose that MutS binds a mismatched primer-template complex, thereby preventing the approach of DNA polymerase to the 3' end of the primer. Our simple methodology improves the efficiency and accuracy of DNA amplification and should therefore benefit various PCR-based applications, ranging from basic biological research to applied medical science.

Recent progress in experimental studies on the catalytic mechanism of cytochrome c oxidase.

Cytochrome c oxidase (CcO) reduces molecular oxygen (O2) to water, coupled with a proton pump from the N-side to the P-side, by receiving four electrons sequentially from the P-side to the O2-reduction site-including Fea3 and CuB-via the two low potential metal sites; CuA and Fea. The catalytic cycle includes six intermediates as follows, R (Fea3 2+, CuB 1+, Tyr244OH), A (Fea3 2+-O2, CuB 1+, Tyr244OH), Pm (Fea3 4+ = O2-, CuB 2+-OH-, Tyr244O•), F (Fea3 4+ = O2-, CuB 2+-OH-, Tyr244OH), O (Fea3 3+-OH-, CuB 2+-OH-, Tyr244OH), and E (Fea3 3+-OH-, CuB 1+-H2O, Tyr244OH). CcO has three proton conducting pathways, D, K, and H. The D and K pathways connect the N-side surface with the O2-reduction site, while the H-pathway is located across the protein from the N-side to the P-side. The proton pump is driven by electrostatic interactions between the protons to be pumped and the net positive charges created during the O2 reduction. Two different proton pump proposals, each including either the D-pathway or H-pathway as the proton pumping site, were proposed approximately 30 years ago and continue to be under serious debate. In our view, the progress in understanding the reaction mechanism of CcO has been critically rate-limited by the resolution of its X-ray crystallographic structure. The improvement of the resolutions of the oxidized/reduced bovine CcO up to 1.5/1.6 Å resolution in 2016 provided a breakthrough in the understanding of the reaction mechanism of CcO. In this review, experimental studies on the reaction mechanism of CcO before the appearance of the 1.5/1.6 Å resolution X-ray structures are summarized as a background description. Following the summary, we will review the recent (since 2016) experimental findings which have significantly improved our understanding of the reaction mechanism of CcO including: 1) redox coupled structural changes of bovine CcO; 2) X-ray structures of all six intermediates; 3) spectroscopic findings on the intermediate species including the Tyr244 radical in the Pm form, a peroxide-bound form between the A and Pm forms, and Fr, a one-electron reduced F-form; 4) time resolved X-ray structural changes during the photolysis of CO-bound fully reduced CcO using XFEL; 5) a simulation analysis for the Pm→Pr→F transition.

A novel single-stranded DNA-specific 3'-5' exonuclease, Thermus thermophilus exonuclease I, is involved in several DNA repair pathways.

Single-stranded DNA (ssDNA)-specific exonucleases (ssExos) are expected to be involved in a variety of DNA repair pathways corresponding to their cleavage polarities; however, the relationship between the cleavage polarity and the respective DNA repair pathways is only partially understood. To understand the cellular function of ssExos in DNA repair better, genes encoding ssExos were disrupted in Thermus thermophilus HB8 that seems to have only a single set of 5'-3' and 3'-5' ssExos unlike other model organisms. Disruption of the tthb178 gene, which was expected to encode a 3'-5' ssExo, resulted in significant increase in the sensitivity to H(2)O(2) and frequency of the spontaneous mutation rate, but scarcely affected the sensitivity to ultraviolet (UV) irradiation. In contrast, disruption of the recJ gene, which encodes a 5'-3' ssExo, showed little effect on the sensitivity to H(2)O(2), but caused increased sensitivity to UV irradiation. In vitro characterization revealed that TTHB178 possessed 3'-5' ssExo activity that degraded ssDNAs containing deaminated and methylated bases, but not those containing oxidized bases or abasic sites. Consequently, we concluded that TTHB178 is a novel 3'-5' ssExo that functions in various DNA repair systems in cooperation with or independently of RecJ. We named TTHB178 as T. thermophilus exonuclease I.

Brown adipose tissue dysfunction promotes heart failure via a trimethylamine N-oxide-dependent mechanism.

Low body temperature predicts a poor outcome in patients with heart failure, but the underlying pathological mechanisms and implications are largely unknown. Brown adipose tissue (BAT) was initially characterised as a thermogenic organ, and recent studies have suggested it plays a crucial role in maintaining systemic metabolic health. While these reports suggest a potential link between BAT and heart failure, the potential role of BAT dysfunction in heart failure has not been investigated. Here, we demonstrate that alteration of BAT function contributes to development of heart failure through disorientation in choline metabolism. Thoracic aortic constriction (TAC) or myocardial infarction (MI) reduced the thermogenic capacity of BAT in mice, leading to significant reduction of body temperature with cold exposure. BAT became hypoxic with TAC or MI, and hypoxic stress induced apoptosis of brown adipocytes. Enhancement of BAT function improved thermogenesis and cardiac function in TAC mice. Conversely, systolic function was impaired in a mouse model of genetic BAT dysfunction, in association with a low survival rate after TAC. Metabolomic analysis showed that reduced BAT thermogenesis was associated with elevation of plasma trimethylamine N-oxide (TMAO) levels. Administration of TMAO to mice led to significant reduction of phosphocreatine and ATP levels in cardiac tissue via suppression of mitochondrial complex IV activity. Genetic or pharmacological inhibition of flavin-containing monooxygenase reduced the plasma TMAO level in mice, and improved cardiac dysfunction in animals with left ventricular pressure overload. In patients with dilated cardiomyopathy, body temperature was low along with elevation of plasma choline and TMAO levels. These results suggest that maintenance of BAT homeostasis and reducing TMAO production could be potential next-generation therapies for heart failure.

Boosting Auto-Induction of Recombinant Proteins in Escherichia coli with Glucose and Lactose Additives.

BACKGROUND: Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level. OBJECTIVES: In this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium. METHODS: First, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7 lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7 lac promoter-based expression). RESULTS: Auto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture. CONCLUSION: The medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.

Time-resolved studies of metalloproteins using X-ray free electron laser radiation at SACLA.

BACKGROUND: The invention of the X-ray free-electron laser (XFEL) has provided unprecedented new opportunities for structural biology. The advantage of XFEL is an intense pulse of X-rays and a very short pulse duration (<10 fs) promising a damage-free and time-resolved crystallography approach. SCOPE OF REVIEW: Recent time-resolved crystallographic analyses in XFEL facility SACLA are reviewed. Specifically, metalloproteins involved in the essential reactions of bioenergy conversion including photosystem II, cytochrome c oxidase and nitric oxide reductase are described. MAJOR CONCLUSIONS: XFEL with pump-probe techniques successfully visualized the process of the reaction and the dynamics of a protein. Since the active center of metalloproteins is very sensitive to the X-ray radiation, damage-free structures obtained by XFEL are essential to draw mechanistic conclusions. Methods and tools for sample delivery and reaction initiation are key for successful measurement of the time-resolved data. GENERAL SIGNIFICANCE: XFEL is at the center of approaches to gain insight into complex mechanism of structural dynamics and the reactions catalyzed by biological macromolecules. Further development has been carried out to expand the application of time-resolved X-ray crystallography. This article is part of a Special Issue entitled Novel measurement techniques for visualizing 'live' protein molecules.

Development of a deep learning-based method to identify "good" regions of a cryo-electron microscopy grid.

Recent advances in cryo-electron microscopy (cryo-EM) have enabled protein structure determination at atomic resolutions. Cryo-EM specimens are prepared by rapidly freezing a protein solution on a metal grid coated with a holey carbon film; this results in the formation of an ice film on each hole. The thickness of the ice film is a critical factor for high-resolution structure determination; ice that is too thick degrades the contrast of the protein image while ice that is too thin excludes the protein from the hole or denatures the protein. Therefore, trained researchers need to manually select "good" regions with appropriate ice thicknesses for imaging. To reduce the time spent on such tasks, we developed a deep learning program consisting of a "detector" and a "classifier" to identify good regions from low-magnification EM images. In our method, the holes in a low-magnification EM image are detected via a detector, and the ice image on each hole is classified as either good or bad via a classifier. The detector detected more than 95% of the holes regardless of the type of samples. The classifier was trained for different types of samples because the appropriate ice thickness varies between sample types. The accuracies of the classifiers were 93.8% for a soluble protein sample (ß-galactosidase) and 95.3% for a membrane protein sample (bovine heart cytochrome c oxidase). In addition, we found that a training data set containing ~ 2100 hole images from 300 low-magnification EM images was sufficient to obtain good accuracy, such as higher than 90%. We expect that the throughput of the cryo-EM data collection step will be greatly improved by using our method.

A temperature-controlled cold-gas humidifier and its application to protein crystals with the humid-air and glue-coating method.

The room-temperature experiment has been revisited for macromolecular crystallography. Despite being limited by radiation damage, such experiments reveal structural differences depending on temperature, and it is expected that they will be able to probe structures that are physiologically alive. For such experiments, the humid-air and glue-coating (HAG) method for humidity-controlled experiments is proposed. The HAG method improves the stability of most crystals in capillary-free experiments and is applicable at both cryogenic and ambient temperatures. To expand the thermal versatility of the HAG method, a new humidifier and a protein-crystal-handling workbench have been developed. The devices provide temperatures down to 4°C and successfully maintain growth at that temperature of bovine cytochrome c oxidase crystals, which are highly sensitive to temperature variation. Hence, the humidifier and protein-crystal-handling workbench have proved useful for temperature-sensitive samples and will help reveal temperature-dependent variations in protein structures.

Structure of bovine cytochrome c oxidase in the ligand-free reduced state at neutral pH.

Cytochrome c oxidase (CcO), the terminal oxidase in cellular respiration, couples proton pumping to O2 reduction. Mammalian CcO resides in the inner mitochondrial membrane. Previously, a model of H-pathway proton pumping was proposed based on various CcO crystal structures. However, all previously determined structures were solved using crystals obtained at pH 5.7, which differs from the environmental pH of CcO in the inner membrane. The structures of fully oxidized and ligand-free reduced CcO at pH 7.3 have now been determined. Structural comparison between the oxidized and reduced states revealed that the structural alterations that occurred upon redox change at pH 5.7 in Asp51, the magnesium-containing cluster, haem groups and helix X, which provide important structural evidence for the H-pathway proton-pumping proposal, also occur at pH 7.3. These structural alterations were restricted to a local region of CcO; no domain movement was detected, nor were significant structural alterations detected in peripheral regions at either pH value. These observations indicate that the small and precise structural alterations that occur over the course of the reaction cycle are not affected by pH change, and that isolated CcO precisely performs proton pumping via the H-pathway over a wide pH range. Because the pH is not uniform across the molecular surface of CcO, the fact that the overall structure of CcO is not affected by pH changes ensures the high enzymatic efficiency of this protein in the mitochondria.