Work

EPR Evidence of Cyanide Binding to the Mn(Mg) Center of Cytochrome c Oxidase: Support for CuA−Mg Involvement in Proton Pumping

Year: 2008

Type: article

Abstract: We examined the anion binding behavior of the Mg(Mn) site in cytochrome c oxidase to test a possible role of this center in proton pumping. Rhodobacter sphaeroides grown in a Mn(II)-rich medium replaces the intrinsic Mg(II) ion with an EPR-detectable Mn(II) ion without change in activity. Due to its close proximity and a shared ligand, oxidized Cu(A) is spin-coupled to the Mn(II) ion, affecting the EPR spectrum. An examination of both bovine and R.s. oxidase crystal structures reveals a hydrogen-bonding pattern in the vicinity of the Mg(II) site that is consistent with three water ligands of the Mg(Mn) center when Cu(A) is oxidized. In the reduced structure, one water molecule in the vicinity of the Cu(A) ligand, E198, moves closer, appearing to be converted into an ionically bonded hydronium ion, while a second water molecule bonded to Mg(Mn) shows evidence of conversion to a hydroxide. The implied proton movement is proposed to be part of a redox-linked export of a pumped proton from the binuclear center into the exit pathway. To test the model, cyanide and azide were added to the oxidized and reduced forms of the enzyme, and Mn(II) CW-EPR and ESEEM spectra were recorded. Addition of azide broadened the CW-EPR spectra for both oxidized and reduced enzyme. Cyanide addition affected the Mn(II) CW-EPR spectrum of reduced cytochrome c oxidase by increasing Mn(II) zero field splitting and broadening the spectral line shapes but had no effect on oxidized enzyme. ESEEM measurements support a differential ability of Mn(II) to bind cyanide in the reduced state of cytochrome c oxidase. This new observation of anion binding at the Mg/Mn site is of interest in terms of accessibility of the buried site and its potential role in redox-dependent proton pumping.

Source: Biochemistry

DOI: 10.1021/bi801391r

Authors: Martyn A. Sharpe, Matthew D. Krzyaniak, Shujuan Xu, John McCracken, Shelagh Ferguson-Miller

Institution: Michigan State University

Language: English

Topic: Metal-Catalyzed Oxygenation Mechanisms

Subfield: Inorganic Chemistry

Field: Chemistry

Domain: Physical Sciences

Open Access status: green

Funder: National Institute of General Medical Sciences

Awards: ENERGY TRANSDUCTION IN CYTOCHROME OXIDASE, G1257321929, Advanced EPR Studies of Radicals in Biological Catalysis, G748428532, Energy Transduction in Cytochrome Oxidase