Electron spin resonance studies on neutrophil cytochrome b558. Evidence that low-spin heme iron is essential for O2-. generating activity.
Cytochrome b558 purified from pig neutrophils was studied to characterize the spin state of the heme iron in relation to its O2-. generating activity. ESR spectra of cytochrome b558 either from resting or stimulated neutrophils showed a low-spin hemoprotein with g1,2,3 of 3.2, 2.1, and 1.3 (estimated). At physiological pH, the oxidized cytochrome b558 is in a purely low-spin state. On lowering or raising pH from 7, the spin state changes to high-spin. The ESR spectrum of high-spin cytochrome b558 was identical to that of methemoglobin, suggesting that the axial-ligand type in both hemoproteins may be the same, i.e. histidine is the fifth ligand. The ratio of the low-spin to high-spin heme in cytochrome b558 was evaluated by magnetic circular dichroism spectroscopy. The pH of cytochrome b558 was varied to form different ratios of the low-spin to high-spin states of the heme, and its O2-. generating activity was examined in cell-free systems. O2-. forming activity decreased concomitant with loss of the low-spin heme, which provides direct evidence that the low-spin state of cytochrome b558 is essential to generate O2-. and the heme retains the low-spin state through the redox cycle.
Fujii
H
H
Department of Inflammation Research, Tokyo Metropolitan Institute of Medical Science (Rinshoken), Japan.
Johnson
M K
MK
Finnegan
M G
MG
Miki
T
T
Yoshida
L S
LS
Kakinuma
K
K
D000818:Animals; D003080:Cold Temperature; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008566:Membranes / Q000737:chemistry / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D018375:Neutrophil Activation; D013053:Spectrophotometry; D013552:Swine
D003573:Cytochrome b Group; D005609:Free Radicals; D008024:Ligands; D006418:Heme; C036888:cytochrome b558; D007501:Iron; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases