title: Surface proton donors for the D-pathway of cytochrome c oxidase in the absence of subunit III.
abstract: The major proton-transfer pathway into the buried active site of cytochrome c oxidase (CcO) is the D-pathway that begins with the subunit I residue Asp-132 on the inner protein surface (the cytoplasmic surface of the aa3-type CcO of Rhodobacter sphaeroides). Asp-132 is surrounded by residues from both subunits I and III. In the absence of subunit III, CcO retains activity, but the functional characteristics of the D-pathway are significantly altered such that the transfer of protons from Asp-132 into the pathway becomes the rate-limiting step. Determination of the pH-dependence of the rate constant for D-pathway proton uptake during the single-turnover of CcO indicates that the pKa of Asp-132 in the absence of subunit III is approximately 7. The removal of subunit III also allows for alternative surface proton donor/acceptors other than Asp-132. With Asp-132 altered to alanine, the rate constant for D-pathway proton uptake is very slow (5 s(-1)) in the presence of subunit III. Once subunit III is removed, the proton uptake rate constant increases 80-fold, to 400 s(-1). The pKa associated with this uptake is >10, and the initial proton donor/acceptor in D132A III (-) is proposed to be a water of the D-pathway rather than an amino acid residue. Arachidonic acid (Aa), which stimulates the activity of several D-pathway mutant CcOs, appears to become the initial proton donor/acceptor in the absence of subunit III, whether or not Asp-132 is altered. Aa shifts the pKa of the initial proton donor to 7.6 for both wild-type (WT) III (-) and D132A III (-). The results indicate that subunit III creates a barrier that helps prevent protons from donors other than Asp-132 from directly accessing the internal waters of the D-pathway, while the subunit also provides an environment that increases the rate at which Asp-132 transfers protons into the D-pathway.
authors.affiliation: Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden. piaa@dbb.su.se
authors.forename: Pia
authors.initials: P
authors.lastname: Adelroth
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hosler
mesh_terms: D016718:Arachidonic Acid / Q000494:pharmacology; D001216:Asparagine / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D021122:Protein Subunits; D011522:Protons; D016718:Arachidonic Acid; D001216:Asparagine; D003576:Electron Transport Complex IV; D010100:Oxygen