Characterization of the Exchangeable Protons in the Immediate Vicinity of the Semiquinone Radical at the QH Site of the Cytochrome bo3 from Escherichia coli

Lai Lai Yap,Rimma I Samoilova,Robert B Gennis,Sergei A Dikanov

doi:10.1074/jbc.m602544200

Abstract

The cytochrome bo3 ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O2 to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. Two-dimensional electron spin echo envelope modulation has been applied to explore the exchangeable protons involved in hydrogen bonding to the semiquinone by substitution of 1H2O by 2H2O. Three exchangeable protons possessing different isotropic and anisotropic hyperfine couplings were identified. The strength of the hyperfine interaction with one proton suggests a significant covalent O-H binding of carbonyl oxygen O1 that is a characteristic of a neutral radical, an assignment that is also supported by the unusually large hyperfine coupling to the methyl protons. The second proton with a large anisotropic coupling also forms a strong hydrogen bond with a carbonyl oxygen. This second hydrogen bond, which has a significant out-of-plane character, is from an NH2 or NH nitrogen, probably from an arginine (Arg-71) known to be in the quinone binding site. Assignment of the third exchangeable proton with smaller anisotropic coupling is more ambiguous, but it is clearly not involved in a direct hydrogen bond with either of the carbonyl oxygens. The results support a model that the semiquinone is bound to the protein in a very asymmetric manner by two strong hydrogen bonds from Asp-75 and Arg-71 to the O1 carbonyl, while the O4 carbonyl is not hydrogen-bonded to the protein.

Highlights

Low affinity site (QL) where the substrate quinol is oxidized and the product is released, and a high affinity site where the bound quinone species acts as a conduit for electrons, similar to the role of the QA site in the bacterial reaction center (3–7, 9)
EPR and ESEEM Spectra of the Semiquinone—The field-sweep ESE spectrum of cyt bo3 recorded at 50 K (Fig. 1) shows only one line from the SQ with g ϳ 2.0047 and the width ϳ1.2 millitesla at the half-height
A Neutral versus an Anionic Semiquinone—The hyperfine couplings for both the non-exchangeable methyl protons as well for the exchangeable protons obtained from HYSCORE and electronnuclear double resonance (ENDOR) experiments provide several important clues about the nature of the SQ species in cyt bo3

Summary

Introduction

Low affinity site (QL) where the substrate quinol is oxidized and the product is released, and a high affinity site where the bound quinone species acts as a conduit for electrons, similar to the role of the QA site in the bacterial reaction center (3–7, 9). The function of the quinone cofactor bound at the QH site as a twoelectron/one-electron transformer is supported by the electrochemical studies, which show that the QH site stabilizes the SQ form of the quinone bound at this site. The x-ray structure of cyt bo (12) does not contain any bound quinone, but site-directed mutagenesis studies (12–15) have identified residues that influence the SQ, resulting in a model for the QH binding site (12). This model (12) proposes hydrogen bonds from Asp-75 and

Results

Discussion

Conclusion