ИСТИНА

Cytochrome bd is a prokaryotic respiratory quinol oxidase identified in a number of pathogens, that is preferentially expressed under low O2 tension or other “stress” conditions. The enzyme couples the exergonic reduction of O2 to 2 H2O to proton motive force generation by transmembrane charge separation. Apart from its role in cell bioenergetics, evidence suggests that cytochrome bd accomplishes a number of additional functions of physiological relevance for the bacterial cell, being seemingly implicated also in microbial resistance to NO-stress and, in general, adaptation to the hostile conditions created by host immunity during the infection process. Cytochrome bd from Escherichia coli contains three hemes, hemes b558 and b595 and heme d, where O2 chemistry takes place through sequential formation of a few catalytic intermediates. Here, the isolated cytochrome bd was investigated by stopped-flow multiwavelength absorption spectroscopy with the aim of measuring the occupancy of the catalytic intermediates at steady-state. We found that, under turnover conditions sustained by dithiothreitol-reduced ubiquinone and O2, the ferryl and oxy-ferrous species are the mostly populated catalytic intermediates, with a minor fraction of the enzyme containing ferric heme d and possibly reduced heme b558. These new findings differ from those obtained with mammalian cytochrome c oxidase, where oxygen intermediates were not found to be populated at detectable levels under similar conditions.The results are discussed in the light of previously proposed models of the cytochrome bd catalytic cycle.