A role for the Cu A–Mg site in pumping would suggest that the Cu A redox potential should have a pH dependence, but the relationship between the redox potential of Cu A and the bulk pH is quite complex. Oxidases that contain a Cu A–Mg site are part of the superfamily, and the inclusion of Mg(II) appears to aid their efficiency i.e., the proton pumping stoichiometry of Cu A–Mg-type oxidases appears to be closer to one than in other types ( 10- 12). Herein, we test a model of how the Mg/Mn site in all eukaryotic and some bacterial heme–copper oxidases may augment the proton pumping mechanism ( 9). A recent examination of high-resolution structures has led us to a focus on the role of water in the facilitation of proton movement ( 8). Despite an extensive body of work using site-directed mutagenesis to examine enzymatic function in detail and the availability of a number of crystal structures, no model has been able to explain how all the members of this structurally diverse family of oxidases are able to pump protons ( 3- 7). This new observation of anion binding at the Mg/Mn site is of interest in terms of accessibility of the buried site and its potential role in redox-dependent proton pumping.Ĭytochrome c oxidase is a proton pump, and this pumping activity is a property of other members of the heme–copper oxidase superfamily ( 1, 2). ESEEM measurements support a differential ability of Mn(II) to bind cyanide in the reduced state of cytochrome c oxidase. Cyanide addition affected the Mn(II) CW-EPR spectrum of reduced cytochrome c oxidase by increasing Mn(II) zero field splitting and broadening the spectral line shapes but had no effect on oxidized enzyme. Addition of azide broadened the CW-EPR spectra for both oxidized and reduced enzyme. To test the model, cyanide and azide were added to the oxidized and reduced forms of the enzyme, and Mn(II) CW-EPR and ESEEM spectra were recorded. The implied proton movement is proposed to be part of a redox-linked export of a pumped proton from the binuclear center into the exit pathway. In the reduced structure, one water molecule in the vicinity of the Cu A ligand, E198, moves closer, appearing to be converted into an ionically bonded hydronium ion, while a second water molecule bonded to Mg(Mn) shows evidence of conversion to a hydroxide. oxidase crystal structures reveals a hydrogen-bonding pattern in the vicinity of the Mg(II) site that is consistent with three water ligands of the Mg(Mn) center when Cu A is oxidized. Due to its close proximity and a shared ligand, oxidized Cu A is spin-coupled to the Mn(II) ion, affecting the EPR spectrum.
Rhodobacter sphaeroides grown in a Mn(II)-rich medium replaces the intrinsic Mg(II) ion with an EPR-detectable Mn(II) ion without change in activity. We examined the anion binding behavior of the Mg(Mn) site in cytochrome c oxidase to test a possible role of this center in proton pumping.
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