Characterization of the semiquinone radical stabilized by the cytochrome aa3-600 menaquinol oxidase of Bacillus subtilis

Abstract

Cytochrome aa3-600 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 O2 to 2H2O. Cytochrome aa3-600 is of interest because it is a very close homologue of the cytochrome bo3 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 bo3 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 aa3-600. Cytochrome aa3-600 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 aa3-600 in comparison with the ubi-semiquinone in cytochrome bo3. Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa3-600 in comparison with ubiquinone in cytochrome bo3. In addition, the electronic structure of the semiquinone cyt aa3-600 is more shifted toward the anionic form from the neutral state in cyt bo3.