The Roles of the Highly Conserved Amino Acid Residues Val236 and Gly232 in the Ligand Channel of Ba3 Cytochrome C Oxidase from Thermus Thermophilus

Abstract

Cytochrome ba3 in the plasma membrane of Thermus thermophilus is a member of the heme-copper oxidase superfamily and functions as a terminal oxidase for aerobic metabolism under limited oxygen concentration. The X-ray crystal structure of Thermus thermophilusba3 (Tt ba3) oxidase revealed a Y-shaped hydrophobic channel leading to the binuclear active site, which is proposed to provide access of ligands such as O2, NO and CO. Recent time-resolved optical absorption (TROA) studies in our laboratory showed that O2 and NO binding is 10-times faster in ba3 than in bovine aa3 and that a tryptophan constriction residue which is present in the ligand channel of the aa3 oxidases, but not in Tt ba3, significantly impeded ligand access in the former (Einarsdottir et al. BBA (2012) 1817, 672-79; McDonald et al. Biochemistry (2013) 52, 640-53). These structural differences could be due to the adaptation of T. thermophilus to microaerobic environments. Comparison of amino acid sequences and crystal structures of the heme-copper oxidases have also identified strictly conserved residues in the ligand channels of these enzymes, including V236 and G232 (Tt ba3 numbering). V236 is located in the proximity of the binuclear active site, and the G232 residue has been postulated to create a small passage-way for ligands to enter the active site. In this study, we investigated by TROA spectroscopy the O2 and NO binding in various Tt ba3 V236 mutants and the mutation of G232 to a larger valine to clarify the role of these conserved amino acid residues in modulating ligand access to the active site in Tt ba3.