Definition of the catalytic site of cytochrome c oxidase: specific ligands of heme a and the heme a3-CuB center.

Abstract

The three-subunit aa3-type cytochrome c oxidase (EC 1.9.3.1) of Rhodobacter sphaeroides is structurally and functionally homologous to the more complex mitochondrial oxidase. The largest subunit, subunit I, is highly conserved and predicted to contain 12 transmembrane segments that provide all the ligands for three of the four metal centers: heme a, heme a3, and CUB. A variety of spectroscopic techniques identify these ligands as histidines. We have used site-directed mutagenesis to change all the conserved histidines within subunit I of cytochrome c oxidase from Rb. sphaeroides. Analysis of the membrane-bound and purified mutant proteins by optical absorption and resonance Raman spectroscopy indicates that His-102 and His-421 are the ligands of heme a, while His-284, His-333, His-334, and His-419 ligate the heme ar-CuB center. To satisfy this ligation assignment, helices II, VI, VII, and X, which contain these histidine residues, must be in close proximity. These data provide empirical evidence regarding the three-dimensional protein structure at the catalytic core of cytochrome c oxidase.Cytochrome c oxidase (EC 1.9.3.1) catalyzes the reduction of oxygen to water at the terminus of the mitochondrial respiratory chain, the principal energy-generating system of eukaryotic organisms (for recent reviews see refs. 1 and 2). Energy conservation is achieved by the coupling of electron transfer through the heme and copper metal centers to proton translocation across the membrane (3). To understand this coupling process it is essential to have a detailed description of the protein structures that form the heme-and copperbinding sites.A number of bacteria synthesize cytochrome c oxidases that are simpler in structure but functionally homologous to the mitochondrial enzymes (2, 4, 5). The aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has recently been purified as a complex of three subunits that are homologues ofthe three mitochondrially encoded subunits of the eukaryotic oxidase (J.P.H. and S.F.-M., unpublished results). The Rb. sphaeroides oxidase has remarkably high turnover (Vm > 1800 sec-1), pumps protons efficiently (0.7 H+/e-) in reconstituted proteoliposomes, and is spectroscopically similar to beef heart cytochrome c oxidase. This preparation clearly establishes the oxidase from Rb. sphaeroides as an excellent bacterial model for examining the structural basis of the catalytic function of the more complex eukaryotic enzyme. The utility of Rb. sphaeroides as a genetic system for analyzing structure/function relationships has been established in previous studies ofthe photosynthetic reaction center (6) and the cytochrome bc complex (7).The genes coding for the three subunits of the Rb. sphaeroides aa3-type oxidase have been cloned and sequenced (refs. 8 and 9 and unpublished data). The genetic organization and deduced amino acid sequences are very similar to those from the closely related bacterium Paracoccus denitrificans (2, 10-12). In addition, subunit I shows 50% sequence identity to subun...

A Second Terminal Oxidase in Sulfolobus acidocaldarius

“…5) are found within hydrophobic regions. The conserved histidines in the transmembrane segments 11, VI, VII and X are ligands of Cu, and the haem irons Minagawa et al, 1992;Shapleigh et al, 1992a;Brown et al, 1993;Calhoun et al, 1993;Hosler et al, 1993). Two characteristic carboxylic acids are marked with arrows in the subunit 111 part (Fig.…”

Ethanol withdrawal posttranslationally decreases the activity of cytochrome c oxidase in an estrogen reversible manner

“…The subunit I is encoded by mitochondrial DNA [12], whereas the subunit IV is encoded by nuclear DNA. In addition, the two subunits interact with each other such that the subunit IV stabilizes the copper center in the subunit I [30,31,32]. A previous study reported that the COX IV formed an adduct with 4-hydroxynonenal (product of lipid peroxidation) after ethanol consumption in rats [33], suggesting a vulnerability of this subunit to ethanol stress.…”

Identification of tissue‐specific isoforms for subunits Vb and VIIa of cytochrome c oxidase isolated from rainbow trout

“…The three largest subunits are encoded by mitochondrial DNA and carry the catalytic centers of the enzyme consisting of heme a, heme a, and Cu, in subunit I [5], and a bimetallic center of two Cu atoms (Cu,) in subunit I1 [6]. Some of the nuclear-coded subunits are expressed as tissue-specific isoforms.…”