Two sites of interaction of anions with cytochrome a in oxidized bovine cytochrome c oxidase.
An interaction between cytochrome a in oxidized cytochrome c oxidase (CcO) and anions has been characterized by EPR spectroscopy. Those anions that affect the EPR g = 3 signal of cytochrome a can be divided into two groups. One group consists of halides (Cl-, Br-, and I-) and induces an upfield shift of the g = 3 signal. Nitrogen-containing anions (CN-, NO2-, N3-, NO3-) are in the second group and shift the g = 3 signal downfield. The shifts in the EPR spectrum of CcO are unrelated to ligand binding to the binuclear center. The binding properties of one representative from each group, azide and chloride, were characterized in detail. The dependence of the shift on chloride concentration is consistent with a single binding site in the isolated oxidized enzyme with a Kd of approximately 3 mm. In mitochondria, the apparent Kd was found to be about four times larger than that of the isolated enzyme. The data indicate it is the chloride anion that is bound to CcO, and there is a hydrophilic size-selective access channel to this site from the cytosolic side of the mitochondrial membrane. An observed competition between azide and chloride is interpreted by azide binding to three sites: two that are apparent in the x-ray structure plus the chloride-binding site. It is suggested that either Mg2+ or Arg-438/Arg-439 is the chloride-binding site, and a mechanism for the ligand-induced shift of the g = 3 signal is proposed.
Fabian
Marian
M
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, USA. fabian@rice.edu
Jancura
Daniel
D
Palmer
Graham
G
D000818:Animals; D000838:Anions / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D045264:Cytochromes a / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D011485:Protein Binding
D000838:Anions; D045264:Cytochromes a; D003576:Electron Transport Complex IV