Two Sites of Interaction of Anions with Cytochrome a in Oxidized Bovine Cytochrome c Oxidase

Marian Fabian,Daniel Jancura,Graham Palmer

doi:10.1074/jbc.m311834200

Abstract

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.

Highlights

electron transfer (ET) is coupled to the generation of transmembrane proton gradient
The complex between cytochrome c and cytochrome c oxidase (CcO) prepared by stoichiometric addition of oxidized cytochrome c to CcO at low ionic strength (10 mM Hepes, pH 7.6, 0.1% Triton X-100) did not affect the peak at g ϭ 3
Identification of the Chloride-binding Site—In the following discussion we will focus on the binding of chloride to cytochrome oxidase that occurs over and above that at the binuclear center, which we and others have characterized in some detail [12, 25, 26, 29, 30]

Summary

The abbreviations used are

CcO, cytochrome c oxidase; CcO1⁄7CN, complex of oxidized CcO with cyanide; ET, electron transfer; DM, n-dodecyl-␤-D-maltoside; Mes, 2-(N-morpholino)ethanesulfonic acid; Ches, 2-(cyclohexylamino)ethanesulfonic acid. Our previous work on ligand binding suggested the interaction of cytochrome a with azide is not mediated by the cytochrome a3-CuB center and that there has to be at least one additional binding site [13]. This site was recently located in the crystal structure of the enzyme-azide complex and shown to be in the proximity of cytochrome a on the hydrophobic surface of the enzyme within the membrane [14]. A detailed characterization of the interaction with chloride and azide shows there is a novel chloride-binding site in native oxidized CcO

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION