Eukaryotic cytochrome c oxidase (CcO) and homologous prokaryotic forms of Rhodobacter and Paraccocus differ in the EPR spectrum of heme a. It was noted that a histidine ligand of heme a (H102) is hydrogen bonded to serine in Rhodobacter (S44) and Paraccocus CcOs, in contrast to glycine in the bovine enzyme. Mutation of S44 to glycine shifts the heme a EPR signal from g z = 2.82 to 2.86, closer to bovine heme a at 3.03, without modifying other properties. Mutation to aspartate, however, results in an oppositely shifted and split heme a EPR signal of g z = 2.72/2.78, accompanied by lower activity and drastically inhibited intrinsic electron transfer from Cu A to heme a. This intrinsic rate is biphasic; the proportion that is slow is pH dependent, as is the relative intensity of the two EPR signal components. At pH 8, the heme a EPR signal at 2.72 is most intense, and the electron transfer rate (Cu A to heme a) is 10-130 s −1 , compared to wild-type at 90000 s −1 . At pH 5.5, the signal at 2.78 is intensified, and a biphasic rate is observed, 50% fast (~wild type) and 50% slow (90 s −1 ). The data support the prediction that the hydrogen-bonding partner of the histidine ligand of heme a is one determinant of the EPR spectral difference between bovine and bacterial CcO. We further demonstrate that the heme a redox potential can be dramatically altered by a nearby carboxyl, whose protonation leads to a proton-coupled electron transfer process.Cytochrome c oxidase (CcO) 1 is the terminal electron acceptor in the respiratory chain of mammalian mitochondria. It contains multiple redox-active metal centers through which electrons are transferred from the soluble electron donor cytochrome c (Cc) to oxygen. The initial electron acceptor in CcO is a dinuclear Cu A in subunit II (Figure 1). The electron is transferred next to heme a, a low-spin six-coordinated heme with two axial histidine ligands (H102 and H421) and then to Cu B and heme a 3 (with one axial ligand, H419); the latter pair make up the active site where oxygen is reduced to water. The movement of electrons through † Supported by National Institutes of Health Grants GM26916 (S.F.-M.), GM54065 (J.M.), and GM20488 (F.M. and B.D.) and NCRR COBRE P20 RR15569 (F.M. and B.D.).*To whom correspondence should be addressed. S.F.-M: telephone, (517) 353-0199; fax, (517) 353-9334; e-mail, fergus20@msu.edu. F.M.: telephone, (479) 575-4999; fax, (479) 575-4049; e-mail, millett@uark.edu. 1 Abbreviations: CcO, cytochrome c oxidase; Cc, cytochrome c; COV, cytochrome c oxidase in phospholipid vesicle (proteoliposome); 3CP, 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy free radical; DEAE, diethylaminoethyl; EPR, electron paramagnetic resonance; LM, lauryl maltoside; Pd, Paracoccus denitrificans; PCR, polymerase chain reaction; Rs, Rhodobacter sphaeroides; RCR, respiratory control ratio; RuCc, ruthenium complex-labeled cytochrome c; Ru 2 Z, ruthenium dimer; TMPD, N,N,N′,N′-tetramethyl-pphenylenediamine. NIH Public Access Author ManuscriptBiochemistry. Author manuscrip...

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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. 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. Due to its close proximity and a shared ligand, oxidized Cu A is spin-coupled to the Mn(II) ion, affecting the EPR spectrum. An examination of both bovine and R.s. 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. 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. 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. 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. Addition of azide broadened the CW-EPR spectra for both oxidized and reduced enzyme. 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. ESEEM measurements support a differential ability of Mn(II) to bind cyanide in the reduced state of cytochrome c oxidase. 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 redoxdependent proton pumping.Cytochrome c oxidase is a proton pump, and this pumping activity is a property of other members of the heme-copper oxidase superfamily (1,2). 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). A recent examination

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