title: Entrance of the proton pathway in cbb3-type heme-copper oxidases.
abstract: Heme-copper oxidases (HCuOs) are the last components of the respiratory chain in mitochondria and many bacteria. They catalyze O(2) reduction and couple it to the maintenance of a proton-motive force across the membrane in which they are embedded. In the mitochondrial-like, A family of HCuOs, there are two well established proton transfer pathways leading from the cytosol to the active site, the D and the K pathways. In the C family (cbb(3)) HCuOs, recent work indicated the use of only one pathway, analogous to the K pathway. In this work, we have studied the functional importance of the suggested entry point of this pathway, the Glu-25 (Rhodobacter sphaeroides cbb(3) numbering) in the accessory subunit CcoP (E25(P)). We show that catalytic turnover is severely slowed in variants lacking the protonatable Glu-25. Furthermore, proton uptake from solution during oxidation of the fully reduced cbb(3) by O(2) is specifically and severely impaired when Glu-25 was exchanged for Ala or Gln, with rate constants 100-500 times slower than in wild type. Thus, our results support the role of E25(P) as the entry point to the proton pathway in cbb(3) and that this pathway is the main proton pathway. This is in contrast to the A-type HCuOs, where the D (and not the K) pathway is used during O(2) reduction. The cbb(3) is in addition to O(2) reduction capable of NO reduction, an activity that was largely retained in the E25(P) variants, consistent with a scenario where NO reduction in cbb(3) uses protons from the periplasmic side of the membrane.
authors: Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
authors: Hyun Ju
authors: HJ
authors: Lee
authors: Robert B
authors: RB
authors: Gennis
authors: Pia
authors: P
authors: Ädelroth
mesh_terms: D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA
chemical_list: D011522:Protons; D009569:Nitric Oxide; D018698:Glutamic Acid; C414443:cbb3 oxidase; D003576:Electron Transport Complex IV; D010100:Oxygen