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J. Biol. Chem., Vol. 278, Issue 34, 31473-31478, August 22, 2003

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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 ¶ and José Edgardo Escamilla  ||

From the Instituto de Fisiología Celular, Facultad de Medicina, and ^¶Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico

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 M_r 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 K_m of 498 µM, a V_max of 21 µmol 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 µM, 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.

Received for publication, March 13, 2003 , and in revised form, June 4, 2003.

^* This work was supported in part by Grants DGAPA-UNAM IN-215801 and CONACYT 34300-N. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Dept. Bioquímica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apdo. Postal 70-242, Mexico City 04510, Mexico. Tel.: 52-55-56225627; Fax: 52-55-56225630; E-mail: eescami{at}ifisiol.unam.mx.

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