Abstract:The ba 3 cytochrome c oxidase from Thermus thermophilus is a B-type oxygen-reducing heme-copper oxidase and a proton pump. It uses only one proton pathway for transfer of protons to the catalytic site, the K B pathway. It was previously shown that the ba 3 oxidase has an overall similar reaction sequence to that in mitochondrial-like A-type oxidases. However, the timing of loading the pump site, and formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, … Show more
“…Readers are referred to both primary and recent review articles from key research groups, summarizing a wide variety of observations and results concerning HCO ligand (CO, NO, and O 2 ) binding dynamics and the O 2 -reduction mechanism across a range of HCOs (and see section 4). 879,963,979,991–999 Some major conclusions are (i) the widely employed method of studying O 2 -binding kinetics by initially photodissociating CO bound to the heme at the heme–Cu binuclear active site is found to be inhibitory in tT ba 3 CcO but not in bovine aa 3 CcO; in other words, the CO flow-flash method does not accurately reflect the O 2 and NO binding kinetics in tT ba 3 under physiological conditions, namely, in the absence of CO. (ii) tT ba 3 CcO possesses a unique Y- shaped extended hydrophobic tunnel as a very low-barrier passage for O 2 or other ligands entering the heme–Cu active site. Here, in tT ba 3 , the binding of dioxygen (and NO (g) ) is “superfast”; they both occur with rate constants of ~10 9 M −1 s −1 , 912,990 near the diffusion limit (and about 100 times greater than that for myoglobin; also see Table 4).…”
Section: Small Molecule Synthetic Models Of Heme-copper Oxidasesmentioning
Heme-copper oxidases (HCOs) are terminal enzymes on the mitochondrial or bacterial respiratory electron transport chain, which utilize a unique heterobinuclear active site to catalyze the 4H+/4e− reduction of dioxygen to water. This process involves a proton-coupled electron transfer (PCET) from a tyrosine (phenolic) residue and additional redox events coupled to transmembrane proton pumping and ATP synthesis. Given that HCOs are large, complex, membrane-bound enzymes, bioinspired synthetic model chemistry is a promising approach to better understand heme-Cu-mediated dioxygen reduction, including the details of proton and electron movements. This review encompasses important aspects of heme-O2 and copper–O2 (bio)chemistries as they relate to the design and interpretation of small molecule model systems and provides perspectives from fundamental coordination chemistry, which can be applied to the understanding of HCO activity. We focus on recent advancements from studies of heme–Cu models, evaluating experimental and computational results, which highlight important fundamental structure–function relationships. Finally, we provide an outlook for future potential contributions from synthetic inorganic chemistry and discuss their implications with relevance to biological O2-reduction.
“…Readers are referred to both primary and recent review articles from key research groups, summarizing a wide variety of observations and results concerning HCO ligand (CO, NO, and O 2 ) binding dynamics and the O 2 -reduction mechanism across a range of HCOs (and see section 4). 879,963,979,991–999 Some major conclusions are (i) the widely employed method of studying O 2 -binding kinetics by initially photodissociating CO bound to the heme at the heme–Cu binuclear active site is found to be inhibitory in tT ba 3 CcO but not in bovine aa 3 CcO; in other words, the CO flow-flash method does not accurately reflect the O 2 and NO binding kinetics in tT ba 3 under physiological conditions, namely, in the absence of CO. (ii) tT ba 3 CcO possesses a unique Y- shaped extended hydrophobic tunnel as a very low-barrier passage for O 2 or other ligands entering the heme–Cu active site. Here, in tT ba 3 , the binding of dioxygen (and NO (g) ) is “superfast”; they both occur with rate constants of ~10 9 M −1 s −1 , 912,990 near the diffusion limit (and about 100 times greater than that for myoglobin; also see Table 4).…”
Section: Small Molecule Synthetic Models Of Heme-copper Oxidasesmentioning
Heme-copper oxidases (HCOs) are terminal enzymes on the mitochondrial or bacterial respiratory electron transport chain, which utilize a unique heterobinuclear active site to catalyze the 4H+/4e− reduction of dioxygen to water. This process involves a proton-coupled electron transfer (PCET) from a tyrosine (phenolic) residue and additional redox events coupled to transmembrane proton pumping and ATP synthesis. Given that HCOs are large, complex, membrane-bound enzymes, bioinspired synthetic model chemistry is a promising approach to better understand heme-Cu-mediated dioxygen reduction, including the details of proton and electron movements. This review encompasses important aspects of heme-O2 and copper–O2 (bio)chemistries as they relate to the design and interpretation of small molecule model systems and provides perspectives from fundamental coordination chemistry, which can be applied to the understanding of HCO activity. We focus on recent advancements from studies of heme–Cu models, evaluating experimental and computational results, which highlight important fundamental structure–function relationships. Finally, we provide an outlook for future potential contributions from synthetic inorganic chemistry and discuss their implications with relevance to biological O2-reduction.
“…The acidic residue (Asp or Glu) at the entrance of the proton pathway is necessary for fast proton uptake in both the A-family D-pathway as well as in the B- and C-family K-pathway analogues [6, 7, 9, 26, 34, 35]. Interestingly, the effects of alteration of N293, located above the entry point of the K C -pathway, appear to be similar to the N139 Rs of the A-type D-pathway in terms of the linkage to internal proton donors (see [36]), although there is no evolutionary relationship.…”
Section: Discussionmentioning
confidence: 99%
“…This form of ‘convergent evolution’ in HCuOs is observed also for the ‘proton-collecting antenna’ around this Glu [39] and the crosslinked Tyr at the active site which is conserved in space but not in sequence [4, 28]. The relative location of the entry-point Glu is different in K B - and K C -pathways, which means that the region above have to be different as well in order to provide connectivity into the subunit I (or CcoN) part of the pathways (discussed also in [35]). The K B and K C pathways are more similar to each other in composition (lacking the lysine, having mostly Tyr and Thr residues) than they are to the K A -pathway, but the only residue conserved between them is the Y227 (see Fig.…”
The heme‑copper oxidases (HCuOs) are terminal components of the respiratory chain, catalyzing oxygen reduction coupled to the generation of a proton motive force. The C-family HCuOs, found in many pathogenic bacteria under low oxygen tension, utilize a single proton uptake pathway to deliver protons both for O reduction and for proton pumping. This pathway, called the K-pathway, starts at Glu-49 in the accessory subunit CcoP, and connects into the catalytic subunit CcoN via the polar residues Tyr-(Y)-227, Asn (N)-293, Ser (S)-244, Tyr (Y)-321 and internal water molecules, and continues to the active site. However, although the residues are known to be functionally important, little is known about the mechanism and dynamics of proton transfer in the K-pathway. Here, we studied variants of Y227, N293 and Y321. Our results show that in the N293L variant, proton-coupled electron transfer is slowed during single-turnover oxygen reduction, and moreover it shows a pH dependence that is not observed in wildtype. This suggests that there is a shift in the pK of an internal proton donor into an experimentally accessible range, from >10 in wildtype to ~8.8 in N293L. Furthermore, we show that there are distinct roles for the conserved Y321 and Y227. In Y321F, proton uptake from bulk solution is greatly impaired, whereas Y227F shows wildtype-like rates and retains ~50% turnover activity. These tyrosines have evolutionary counterparts in the K-pathway of B-family HCuOs, but they do not have the same roles, indicating diversity in the proton transfer dynamics in the HCuO superfamily.
“…four that are pumped and the remaining two protons used for reduction of O 2 to H 2 O at the catalytic site. In the B-type oxidases all protons are transferred through a single pathway, which approximately overlaps in space with the K pathway in the A-type oxidases 3,6,7 . Hence, there are alternative pathways for transfer of the pumped protons from the n -side in different oxidases.Figure 1Structural model.…”
In cytochrome c oxidase (CytcO) reduction of O2 to water is linked to uptake of eight protons from the negative side of the membrane: four are substrate protons used to form water and four are pumped across the membrane. In bacterial oxidases, the substrate protons are taken up through the K and the D proton pathways, while the pumped protons are transferred through the D pathway. On the basis of studies with CytcO isolated from bovine heart mitochondria, it was suggested that in mitochondrial CytcOs the pumped protons are transferred though a third proton pathway, the H pathway, rather than through the D pathway. Here, we studied these reactions in S. cerevisiae CytcO, which serves as a model of the mammalian counterpart. We analyzed the effect of mutations in the D (Asn99Asp and Ile67Asn) and H pathways (Ser382Ala and Ser458Ala) and investigated the kinetics of electron and proton transfer during the reaction of the reduced CytcO with O2. No effects were observed with the H pathway variants while in the D pathway variants the functional effects were similar to those observed with the R. sphaeroides CytcO. The data indicate that the S. cerevisiae CytcO uses the D pathway for proton uptake and presumably also for proton pumping.
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