Abstract

Cytochrome aa(3)-600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O(2) to 2H(2)O. Cytochrome aa(3)-600 is of interest because it is a very close homologue of the cytochrome bo(3) ubiquinol oxidase from Escherichia coli, except that it uses menaquinol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo(3) has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiquinone. The current work initiates studies to characterize the equivalent site in cytochrome aa(3)-600. Cytochrome aa(3)-600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaquinone-7 and that the enzyme stabilizes a mena-semiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa(3)-600 in comparison with the ubi-semiquinone in cytochrome bo(3). Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa(3)-600 in comparison with ubiquinone in cytochrome bo(3). In addition, the electronic structure of the semiquinone cyt aa(3)-600 is more shifted toward the anionic form from the neutral state in cyt bo(3).

Highlights

  • A number of prokaryotes contain heme-copper respiratory oxygen reductases, which utilize a membrane-bound quinol as the substrate [1, 2]

  • There is a homologue of cyt bo3 in B. subtilis called cytochrome aa3-600, and as expected, this enzyme is a menaquinol oxidase (10 –16)

  • The results show a distinctly different pattern of hydrogen bonding between the protein and SQ species in the menaquinol oxidase than that observed with the E. coli cyt bo3 ubiquinol oxidase

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Summary

Introduction

A number of prokaryotes contain heme-copper respiratory oxygen reductases, which utilize a membrane-bound quinol as the substrate (electron donor) [1, 2]. The equivalent nitrogens from Arg-71 and His-98 interact with the SQ in cyt bo3, values of hyperfine couplings are not the same, reflecting differences in hydrogen bond geometry and overlap of the electronic orbitals of the SQ and protein residues.

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