Spectroscopic identification of the heme axial ligation of cytochrome b(558) in the NADPH oxidase of porcine neutrophils

The combination of electron paramagnetic resonance (EPR), near-infrared magnetic circular dichroism (NIR-MCD) and resonance Raman (RR) spectroscopies at cryogenic temperatures has been used to identify the axial heme Ligation of the low spin cytochrome b(558) component of NADPH oxidase from porcine blood neutrophils, The EPR and NIR-MCD results indicate the presence of two distinct forms in frozen solution; one with a low field g-value at 3.23 and porphyrin(pi)-to-Fe(III) charge transfer maximum at 1660 nm and the other a low field g-value at 3.00 and porphyrin(pi)-to-Fe(III) charge transfer maximum at 1510 nm, On the basis of these properties and the RR studies, both are attributed to forms of cytochrome b(558) with bis-histidine axial ligation, The origin of the observed heterogeneity, the location and identity of the specific histidines involved in ligating the heme, and the role of the heme prosthetic group in O-2(-) production are discussed in light of these results.