The Functional Role of Conserved Acidic Residues of the Qcr7 Protein of the Cytochrome bc 1 Complex in Saccharomyces cerevisiae So Young Lee,* , † Sandeep Raha,* , † Bhushan Nagar,† and Brian H. Robinson* , † , ‡ ,1 *Metabolism Research Programme, The Research Institute, †Department of Biochemistry and ‡Department of Paediatrics, University of Toronto and The Research Institute, the Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8 Received April 19, 2001, and in revised form June 15, 2001; published online August 24, 2001 The 14-kDa Qcr7 protein represents one of the 10 subunits that are components of a functional cyto- chrome bc 1 complex in Sacharomyces cerevisiae. Pre- vious studies have shown that the N-terminus of the Qcr7 protein may be involved in the assembly of the cytochrome bc 1 complex and its C-terminus by inter- acting with cytochrome b and QCR8 proteins. It has also been suggested that Qcr7 protein may be involved in proton pumping. The coding sequence for two highly conserved aspartate residues, D46 and D47, in the QCR7 gene was altered by site-directed mutagen- esis and the mutated genes expressed in cells lacking a functional QCR7 gene. Mutants D46E, D46G, D46N, and D47E were comparable to wild type in growth phenotype on nonfermentable carbon sources. Mu- tants D47G and D47N were respiratory deficient and analysis of complex components by immunoblotting and spectral analysis of cytochrome b suggests defec- tive assembly. Despite being respiratory competent and having normal electron transport rates in broken mitochondria, the mutant D46G had markedly re- duced ATP synthesis from electron transport reac- tions catalyzed by complexes II plus III of the respira- tory chain. This suggests that the geometry of proton uptake by the bc 1 complex is disturbed by the muta- tion in D46. © 2001 Academic Press Key Words: Qcr7 protein; bc 1 complex; acidic residues. The cytochrome bc 1 complex (ubiquiniol: cytochrome c oxidoreductase) is one of the multisubunit complexes of the mitochondrial respiratory chain and is embed- ded in the inner membrane of mitochondria (1, 2). X-ray crystallography reveals the pear-shaped dimer of the cytochrome bc 1 complex in bovine and chicken to be anchored by cytochrome b in the membrane (3, 4). The cytochrome bc 1 complex in yeast consists of 10 sub- units, 3 of which contain prosthetic groups which form redox centers. These are cytochrome b, cytochrome c 1 , and the Rieske iron–sulfur protein (5). In addition, there are six additional supernumerary subunits that are essential for normal functioning of bc 1 complex based on gene inactivation studies but their functions are yet to be determined (6 –9). The mechanism of electron transfer coupled to proton translocation by the cytochrome bc 1 complex is explained by the protonmo- tive Q cycle (10). Cytochrome c 1 and iron–sulfur pro- tein are located on the electropositive cytosolic side of the inner membrane of mitochondria, where the iron– sulfur protein accepts the first electron from ubiquinol at the (Q o ) site (11). It is believed that electron transfer to cytochrome b follows, and the deposition of protons on the cytosolic side of the inner mitochondrial mem- brane occur at this Q o site. The reduction of ubiquinone and the entry of protons to reform ubiquinol occur at the negative matrix (Q i ) side of the inner membrane of mitochondria. Recent findings have shown that the mobile part of iron–sulfur protein may be involved in the electron transfer process at the Q o site (4, 12). However, the mechanism of electron transfer at the Q i site of the inner membrane has yet to be elucidated. The Qcr7 protein is encoded by the QCR7 gene and is a 14.5-kDa polypeptide in yeast. It was found to be homologous to the 13.4-kDa subunit of the bovine cy- tochrome bc 1 complex (13–15) and has been implicated 1 To whom correspondence and reprint requests should be ad- dressed at Metabolism Research Programme, the Research Institute, the Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Fax: (416) 813-8700. E-mail: bhr@sickkids.on.ca. 0003-9861/01 $35.00 207 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 393, No. 2, September 15, pp. 207–214, 2001 doi:10.1006/abbi.2001.2497, available online at http://www.idealibrary.com on