Proton-dependent electron transfer from CuA to heme a and altered EPR spectra in mutants close to heme a of cytochrome oxidase

Denise A. Mills, Shujuan Xu, Lois Geren, Carrie Hiser, Ling Qin, Martyn A. Sharpe, John McCracken, Bill Durham, Francis Millett, Shelagh Ferguson-Miller

Research output: Contribution to journalArticle

10 Scopus citations


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 gz = 2.72/2.78, accompanied by lower activity and drastically inhibited intrinsic electron transfer from CuA 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 (CuA 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.

Original languageEnglish (US)
Pages (from-to)11499-11509
Number of pages11
Issue number44
StatePublished - Nov 4 2008

ASJC Scopus subject areas

  • Biochemistry

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