Proton transfer in cytochrome bd-I from E. coli involves Asp-105 in CydB.

Autor: Janczak M; Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden., Vilhjálmsdóttir J; Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden., Ädelroth P; Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden. Electronic address: piaa@dbb.su.se.
Jazyk: angličtina
Zdroj: Biochimica et biophysica acta. Bioenergetics [Biochim Biophys Acta Bioenerg] 2024 Nov 01; Vol. 1865 (4), pp. 149489. Date of Electronic Publication: 2024 Jul 14.
DOI: 10.1016/j.bbabio.2024.149489
Abstrakt: Cytochrome bds are bacterial terminal oxidases expressed under low oxygen conditions, and they are important for the survival of many pathogens and hence potential drug targets. The largest subunit CydA contains the three redox-active cofactors heme b 558 , heme b 595 and the active site heme d. One suggested proton transfer pathway is found at the interface between the CydA and the other major subunit CydB. Here we have studied the O 2 reduction mechanism in E. coli cyt. bd-I using the flow-flash technique and focused on the mechanism, kinetics and pathway for proton transfer. Our results show that the peroxy (P) to ferryl (F) transition, coupled to the oxidation of the low-spin heme b 558 is pH dependent, with a maximum rate constant (~10 4  s -1 ) that is slowed down at higher pH. We assign this behavior to rate-limitation by internal proton transfer from a titratable residue with pK a  ~ 9.7. Proton uptake from solution occurs with the same P➔F rate constant. Site-directed mutagenesis shows significant effects on catalytic turnover in the CydB variants Asp58 B ➔Asn and Asp105 B ➔Asn variants consistent with them playing a role in proton transfer. Furthermore, in the Asp105 B ➔Asn variant, the reactions up to P formation occur essentially as in the wildtype bd-I, but the P➔F transition is specifically inhibited, supporting a direct and specific role for Asp105 B in the functional proton transfer pathway in bd-I. We further discuss the possible identity of the high pK a proton donor, and the conservation pattern of the Asp-105 B in the cyt. bd superfamily.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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