A Novel Double Heme Substitution Produces a Functional bo3 Variant of the Quinol Oxidase aa3 of Bacillus cereus: PURIFICATION AND PARTIAL CHARACTERIZATION

Martha Contreras-Zentella,Guillermo Mendoza,Jorge Membrillo-Hernández,José Edgardo Escamilla

doi:10.1074/jbc.m302583200

Martha Contreras-Zentella, Guillermo Mendoza + Show 2 more

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https://doi.org/10.1074/jbc.m302583200

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Abstract

A novel bo3-type quinol oxidase was highly purified from Bacillus cereus PYM1, a spontaneous mutant unable to synthesize heme A and therefore spectroscopically detectable cytochromes aa3 and caa3. The purified enzyme contained 12.4 nmol of heme O and 11.5 nmol of heme B mg-1 protein. The enzyme was composed of two subunits with an Mr of 51,000 and 30,000, respectively. Both subunits were immunoreactive to antibodies raised against the B cereus aa3 oxidase. Moreover, amino-terminal sequence analysis of the 30-kDa subunit revealed that the first 19 residues were identical to those from the 30-kDa subunit of the B. cereus aa3 oxidase. The purified bo3 oxidase failed to oxidize ferrrocytochrome c (neither yeast nor horse) but oxidized tetrachlorohydroquinol with an apparent Km of 498 microM, a Vmax of 21 micromol of O2 min-1mg-1, and a calculated turnover of 55 s-1. The quinol oxidase activity with tetrachlorohydroquinol was inhibited by potassium cyanide and 2-n-heptyl 4-hydroxyquinoline-N-oxide with an I50 of 24 and 300 microM, respectively. Our results demonstrate that the bo3 oxidase of this mutant is not the product of a new operon but instead is a cytochrome aa3 apoprotein encoded by the qox operon of the aa3 oxidase of B. cereus wild type promiscuously assembled with hemes B and O replacing heme A, producing a novel bo3 cytochrome. This is the first reported example of an enzymatically active promiscuous oxidase resulting from the simultaneous substitution of its original hemes in the high and low spin sites.

Highlights

A novel bo3-type quinol oxidase was highly purified from Bacillus cereus PYM1, a spontaneous mutant unable to synthesize heme A and spectroscopically detectable cytochromes aa3 and caa3
Our results demonstrate that the bo3 oxidase of this mutant is not the product of a new operon but instead is a cytochrome aa3 apoprotein encoded by the qox operon of the aa3 oxidase of B. cereus wild type promiscuously assembled with hemes B and O replacing heme A, producing a novel bo3 cytochrome
The equivalent chromatographic fraction from extracts of the wild type strain of B. cereus contained TMPD oxidase activity, and as reported earlier [22], further purification of this fraction confirmed the presence of cytochrome caa3

Summary

Introduction

A novel bo3-type quinol oxidase was highly purified from Bacillus cereus PYM1, a spontaneous mutant unable to synthesize heme A and spectroscopically detectable cytochromes aa and caa. Heme O is normally not present in the Bacillaceae family; the presence of heme O has been reported in wild type cells growing under limited aeration [3, 4] or in mutants deficient in the ctaA gene that encodes for the oxygenase responsible for the conversion of heme O into heme A (i.e. Bacillus subtilis RB829R [14]) as well as, in other genetically uncharacterized (presumably ctaA) mutants (B. subtilis FG83 [15] and Bacillus cereus PYM1 [16]) In none of these mutants were any type a cytochromes spectroscopically detected; instead, the presence of a functional type o oxidase in membranes was suggested based on a spectral studies of CO-binding and O2 displacement upon photodissociation at subzero temperatures [15, 17]. An enzymatically active promiscuous oxidase bearing double heme substitution has yet to be

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