Insight into the active-site structure and function of cytochrome oxidase by analysis of site-directed mutants of bacterial cytochrome aa 3 and cytochrome bo (original) (raw)
Related papers
Biochemistry, 2009
Properties of Arg481 mutants of the aa 3 -type cytochrome c oxidase from Rhodobacter sphaeroides suggest that neither R481 nor the nearby D-propionate of heme a 3 is likely to be the proton loading site of the proton pump Abstract Cytochrome c oxidase utilizes the energy from electron transfer and reduction of oxygen to water and pumps protons across the membrane, generating a proton motive force. A large body of biochemical work has shown that all the pumped protons enter the enzyme through the D-channel, which is apparent in X-ray structures as a chain of water molecules connecting D132 at the cytoplasmic surface of the enzyme, to E286, near the enzyme active site. The exit pathway utilized by pumped protons beyond this point and leading to the bacterial periplasm is not known. Also not known is the proton loading site (or sites) which undergoes changes in pK a in response to the chemistry at the enzyme active site and drives the proton pump mechanism. In this paper we examine the role of R481, a highly conserved arginine that forms an ion pair with the D-propionate of heme a 3 . The R481H, R481N, R481Q and R481L mutants were examined. The R481H mutant oxidase is about 18% active and pumps protons with about 40% of the stoichiometry of the wild type. The R481N, R481Q and R481L mutants each retain only about 5% of the steady state activity, and this is shown to be due to inhibition of steps in the reaction of O 2 with the reduced enzyme. Neither the R481N nor the R481Q mutant oxidases pump protons but, remarkably, the R481L mutant does pump protons with the same efficiency as the R481H mutant. Since the proton pump is clearly operating in the R481L mutant, these results rule out an essential role in the proton pump mechanism for R481 or its hydrogen bond partner, the D-propionate of heme a 3 .
Biochemistry, 1996
The molecular mechanism by which proton pumping is coupled to electron transfer in cytochrome c oxidase has not yet been determined. However, several models of this process have been proposed which are based on changes occurring in the vicinity of the redox centers of the enzyme. Recently, a model was described in which a well-conserved tyrosine residue in subunit I (Y422) was proposed to undergo ligand exchange with the histidine ligand (H419) of the high-spin heme a 3 during the catalytic cycle, allowing both residues to serve as part of a proton transporting system. Site-directed mutants of Y422 have been constructed in the aa 3-type cytochrome c oxidase of Rhodobacter sphaeroides to test this hypothesis (Y422A, Y422F). The results demonstrate that Y422 is not an essential residue in the electron transfer and proton pumping mechanisms of cytochrome c oxidase. However, the results support the predicted proximity of Y422 to heme a 3 , as now confirmed by crystal structure. In addition, it is shown that the pH-dependent reversed electron transfer between heme a and heme a 3 is normal in the Y422F mutant. Hence, these data also demonstrate that Y422 is not the residue previously postulated to interact electrostatically with heme a 3 , nor is it responsible for the unique EPR characteristics of heme a in this bacterial oxidase.
Proceedings of the National Academy of Sciences, 1997
The crystal structures of cytochrome c oxidase from both bovine and Paracoccus denitrificans reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase. In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome c oxidase from Rhodobacter sphaeroides. A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a 3 (Fe 4؉ ؍ O 2؊ ) to the oxidized form (Fe 3؉ OH ؊ ). This redox step requires the delivery of a ''chemical'' H ؉ to protonate the reduced oxygen atom and is also coupled to proton pumping. It is found that mutations in the K channel (K362M and T359A) have virtually no effect on the ferryl-oxo-to-oxidized (F-to-Ox) transition, although steady-state turnover is severely limited. In contrast, electrogenic proton transfer at this step is strongly suppressed by mutations in the D channel. The results strongly suggest that the functional roles of the two channels are not the separate delivery of chemical or pumped protons, as proposed recently [Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. (1995) Nature (London) 376, 660-669]. The D channel is likely to be involved in the uptake of both ''chemical'' and ''pumped'' protons in the F-to-Ox transition, whereas the K channel is probably idle at this partial reaction and is likely to be used for loading the enzyme with protons at some earlier steps of the catalytic cycle. This conclusion agrees with different redox states of heme a 3 in the K362M and E286Q mutants under aerobic steady-state turnover conditions.
Biochemistry, 1996
Several putative proton transfer pathways have been identified in the recent crystal structures of the cytochrome oxidases from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669] and bovine [Tsukihara (1996) Science 272, 1138-1144]. A series of residues along one face of the amphiphilic transmembrane helix IV lie in one of these proton transfer pathways. The possible role of these residues in proton transfer was examined by site-directed mutagenesis. The three conserved residues of helix IV that have been implicated in the putative proton transfer pathway (Ser-201, Asn-207, and Thr-211) were individually changed to alanine. The mutants were purified, analyzed for steady-state turnover rate and proton pumping efficiency, and structurally probed with resonance Raman spectroscopy and FTIR difference spectroscopy. The mutation of Ser-201 to alanine decreased the enzyme turnover rate by half, and was therefore further characterized using EPR spectroscopy and rapid kinetic methods. The results demonstrate that none of these hydrophilic residues are essential for proton pumping or oxygen reduction activities, and suggest a model of redundant or flexible proton transfer pathways. Whereas previously reported mutants at the start of this putative channel (e.g., Asp-132-Asn) dramatically influence both enzyme turnover and coupling to proton pumping, the current work shows that this is not the case for all residues observed in this channel.
Structural basis for functional properties of cytochromecoxidase
bioRxiv (Cold Spring Harbor Laboratory), 2023
Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes, thereby establishing the proton gradient required for ATP synthesis 1. The full turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized by molecular oxygen to the metastable oxidized OH state, and a reductive phase, in which OH is reduced back to the R state. During each of the two phases, two protons are translocated across the membranes 2. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation 2,3. How the O state structurally differs from OH remains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX) 4 , we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state 5,6 , are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three CuB ligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from OH, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of CcO. Main Mammalian CcO is a large integral membrane protein comprised of 13 subunits. It contains four redox active centers, CuA, heme a, and a heme a3/CuB binuclear center (BNC) (Fig. 1A). Molecular oxygen binds to the heme a3 iron in the BNC, where it is reduced to two water molecules by accepting four electrons from cytochrome c and four protons (the "substrate" protons) from the negative side (N-side) of the mitochondrial membrane (Fig. 1A). The energy derived from the oxygen reduction chemistry is used to drive the translocation of four protons (the "pumped" protons) from the N-side to the positive side (P-side) of the membrane 1,7. Strong evidence suggests that the substrate protons are delivered to the BNC via the D and K-channel (see Extended Data
Biochemistry, 2005
Internal electron transfer (ET) to heme a 3 during anaerobic reduction of oxidized bovine heart cytochrome c oxidase (CcO) was studied under conditions where heme a and Cu A were fully reduced by excess hexaamineruthenium. The data show that ET to heme a 3 is controlled by the state of ionization of a single protolytic residue with a pK a of 6.5 ( 0.2. On the basis of the view that ET to the catalytic site is limited by coupled proton transfer, this pK a was attributed to Glu60 which is located at the entrance of the proton-conducting K channel on the matrix side of CcO. It is proposed that Glu60 controls proton entry into the channel. However, even with this channel open, there is the second factor that regulates ET, and this is ascribed to the rate of proton diffusion in the channel. In addition, it is concluded that proton transfer in the K channel is reversibly inhibited by the detergent Triton X-100. It is also found that the rate of ET to heme a 3 in the as-isolated resting enzyme and in CcO "activated" by reaction of fully reduced enzyme with O 2 is the same, implying that the catalytic sites of these two forms of oxidized enzyme are essentially identical.