A Cytochrome bb′-type Quinol Oxidase inBacillus subtilis Strain 168*

Abstract

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa ₃, which is a cytochromec oxidase, and cytochrome aa ₃ andbd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa ₃ andcaa ₃ terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bdcontent of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a “cytochrome o”-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb′ type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB andythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion ofythAB in strain LUH27 or the presence of theyth genes on plasmid did not affect the expression of thebb′ oxidase. It is concluded that the novelbb′-type oxidase probably is cytochrome bdencoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e.cytochromeb ₅₅₈ b ₅₉₅ b′.