Two different aa3‐type cytochromes can be purified from the bacterium Bacillus cereus

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Abstract
Two aa3‐type cytochromes were purified from membranes of sporulating Bacillus cereus. One of them, an aa3 complex, was found to be composed of two subunits (51 and 31 kDa), two a hemes and three copper atoms, thus being similar to the cytochrome aa3 previously purified from vegetative B. cereus [García‐Horsman, J. A., Barquera, B., González‐Halphen, D. & Escamilla, J. E. (1991) Mol. Microbiol. 5, 197–205]. The second isoform, a caa3 complex, was expressed in sporulating cells only, and was found to be composed of two subunits (51 and 37 kDa). The 37‐kDa subunit (subunit II) is a heme‐c‐containing polypeptide as shown by its peroxidase activity in SDS/PAGE gels and by its spectral features. Both subunits of the caa3 complex immunologically cross‐reacted with antiserum raised against B. cereus cytochrome aa3, suggesting homology between the two enzymes. Also, the heme‐c‐containing subunit of the caa3 complex was reactive with anti‐(bovine cytochrome c) antiserum, but not with anti‐(bovine cytochrome c1) antiserum. In addition to one heme c and two hemes a, the caa3 complex contained three copper atoms. Kinetic comparison of aa3 and caa3 complexes revealed that the latter is slightly more active (k= 150 s−1) and has a lower affinity to yeast cytochrome c (Km= 76 μM) and to oxygen (Km= 2 μM) as compared with cytochrome aa3 (100 s−1, 10 μM, and 5 μM, respectively).