Modulation of the heme environment of neutrophil cytochrome b558 to a "cytochrome P450-like" structure by pyridine.
The effect of pyridine on the heme environment of cytochrome b558 was studied using ESR and optical absorption spectroscopy in relation to the O2(-)-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 b558 shifted which correlated with a concomitant decrease in O2(-)-generating activity. In addition, the g = 3.2 signal of cytochrome b558 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 b558 by shifting the 5th heme ligand (histidine) to a nearby thiolate 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 b558 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 b558 with a g-value of 3.2 is essential to the O2(-)-forming reaction of the NADPH oxidase system. From sequence alignments of cytochrome P450 with those of large and small subunits of cytochrome b558, the heme in cytochrome b558 appears to be specifically associated with the large subunit.
Fujii
H
H
Department of Inflammation Research, Tokyo Metropolitan Institute of Medical Science, Japan.
Yonetani
T
T
Miki
T
T
Kakinuma
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D003573:Cytochrome b Group / Q000097:blood / Q000737:chemistry* / Q000187:drug effects; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000097:blood; D019255:NADPH Oxidases; D009504:Neutrophils / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects*; D011725:Pyridines / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013481:Superoxides / Q000097:blood; D013552:Swine
D003573:Cytochrome b Group; D011725:Pyridines; D013481:Superoxides; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C023666:pyridine