MODULATION OF THE HEME ENVIRONMENT OF NEUTROPHIL CYTOCHROME B(558) TO A CYTOCHROME P450-LIKE STRUCTURE BY PYRIDINE

The effect of pyridine on the heme environment of cytochrome b(558) was studied using ESR and optical absorption spectroscopy in relation to the O-2(-)-generating activity in the NADPH oxidase system of stimulated pig neutrophils. As the concentration of pyridine increased, the absorption maxima of the alpha- and gamma-bands of cytochrome b(558) shifted which correlated with a concomitant decrease in O, generating activity. In addition, the g = 3.2 signal of cytochrome b(558) decreased with the concomitant appearance of a new ESR spectrum that strikingly resembled that of cytochrome P450. The results suggest that pyridine induces a structural modification in the heme environment of cytochrome b(558) by shifting the 5th heme ligand (histidine) to a nearby thio late group without direct binding of pyridine to the heme. The existence of a reactive thiolate near the heme iron was confirmed by pretreatment of blocked cytochrome b(558) with p-chloromercuribenzoate, which completely inhibited the formation of the cytochrome P450-like ESR spectrum. The results provide further evidence that a low-spin heme iron of cytochrome b(558) with a g-value of 3.2 is essential to the O-2(-)-forming reaction of the NADPH oxidase system, From sequence alignments of cytochrome P450 with those of large and small subunits of cytochrome b(558) the heme in cytochrome b(588) appears to be specifically associated with the large subunit.