Exploring by Pulsed EPR the Electronic Structure of Ubisemiquinone Bound at the QH Site of Cytochrome bo3 from Escherichia coli with in Vivo13C-Labeled Methyl and Methoxy Substituents

Myat T Lin,Alexander A Shubin,Rimma I Samoilova,Kuppala V Narasimhulu,Amgalanbaatar Baldansuren,Robert B Gennis,Sergei A Dikanov

doi:10.1074/jbc.m110.206821

Abstract

The cytochrome bo(3) 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 O(2) to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. The semiquinone is also formed in the D75E mutant, where the mutation has little influence on the catalytic activity, and in the D75H mutant, which is virtually inactive. In this work, wild-type cytochrome bo(3) as well as the D75E and D75H mutant proteins were prepared with ubiquinone-8 (13)C-labeled selectively at the methyl and two methoxy groups. This was accomplished by expressing the proteins in a methionine auxotroph in the presence of l-methionine with the side chain methyl group (13)C-labeled. The (13)C-labeled quinone isolated from cytochrome bo(3) was also used for the generation of model anion radicals in alcohol. Two-dimensional pulsed EPR and ENDOR were used for the study of the (13)C methyl and methoxy hyperfine couplings in the semiquinone generated in the three proteins indicated above and in the model system. The data were used to characterize the transferred unpaired spin densities on the methyl and methoxy substituents and the conformations of the methoxy groups. In the wild type and D75E mutant, the constraints on the configurations of the methoxy side chains are similar, but the D75H mutant appears to have altered methoxy configurations, which could be related to the perturbed electron distribution in the semiquinone and the loss of enzymatic activity.

Highlights

A SQ can bind within a protein site in a manner that favors either the neutral (QH1⁄7) or anionic (Q. ) form or intermediate states with partial charge remaining on the SQ
Based on molecular orbital calculations, the influence of the methoxy torsional angle on the reduction potentials of the UQ can be as significant as 0.4 eV, and it has been proposed that electron transfer reactions can be controlled allosterically through specific orientations of the methoxy groups of the UQ imposed by echo envelope modulation; HYSCORE, hyperfine sublevel correlation; ENDOR, electron-nuclear double resonance; mT, millitesla
The hyperfine couplings for the methoxy protons of SQ anion radicals in isopropyl alcohol have been measured by ENDOR spectroscopy, it is difficult to analyze the conformation of the methoxy groups because the methoxy protons are far from the quinone ring and possess only small hyperfine couplings [14]

Summary

Introduction

A SQ can bind within a protein site in a manner that favors either the neutral (QH1⁄7) or anionic (Q. ) form or intermediate states with partial charge remaining on the SQ. The EPR studies of the semiquinone at the QH site have found hyperfine couplings with methyl and exchangeable, hydrogen-bonded protons consistent with a neutral radical species, indicating significant asymmetry in the distribution of the unpaired spin density (24 – 26). We report the selective 13C labeling of the methyl and methoxy substituents of the UQ-8 cofactor in cyt bo3 This has allowed electron spin echo envelope modulation (ESEEM) and electron-nuclear double resonance (ENDOR) measurements of the methyl and methoxy 13C hyperfine couplings of the SQ radical at the QH site of WT and D75E and D75H mutant cyt bo and the UQ-8 anion radical in isopropyl alcohol glassy solution and their analysis for characterization of spin density distribution and conformation of methoxy groups

Results

Discussion

Conclusion