Electron spin resonance studies on neutrophil cytochrome b sub(558). Evidence that low-spin heme iron is essential for oxygen free radical generating activity

Cytochrome b sub(558) purified from pig neutrophils was studied to characterize the spin state of the heme iron in relation to its oxygen free radical generating activity. ESR spectra of cytochrome b sub(558) either from resting or stimulated neutrophils showed a low-spin hemoprotein with g sub(1,2,3) of 3.2, 2.1, and 1.3 (estimated). At physiological pH, the oxidized cytochrome b sub(558) 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 b sub(558) 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 b sub(558) was evaluated by magnetic circular dichroism spectroscopy. The pH of cytochrome b sub(558) was varied to form different ratios of the low-spin to high-spin states of the heme, and its oxygen free radical generating activity was examined in cell-free systems. Oxygen free radical forming activity decreased concomitant with loss of the low-spin heme, which provides direct evidence that the low-spin state of cytochrome b sub(558) is essential to generate oxygen free radical and the heme retains the low-spin state through the redox cycle.