The Active Form of the Ferric Heme in Neutrophil Cytochrome b558 Is Low-Spin in the Reconstituted Cell-Free System in the Presence of Amphophil

The spin state of the heme in superoxide (O2*- )-producing cytochrome b558 purified from pig neutrophils was examined by means of room-temperature magnetic circular dichroism (MCD) under physiological conditions. Cytochrome b558 with varying amounts of low-spin and high-spin heme was prepared by either pH adjustment or heat treatment, and the O2*−-forming activity in a cell-free system was found to correlate with the low-spin heme content. The possibility that the O2*−-forming activity results from a transient high-spin ferric heme form that is induced during activation by anionic amphophils has also been investigated. EPR spectra of cytochrome b558 activated by either arachidonic acid or myristic acid, showed that a transient high-spin ferric species accounting for approximately 50%of the heme appeared in the presence of arachidonic acid, but not in the presence of myristic acid. Hence the appearance of a transient high-spin ferric heme species on activation with an amphophil does not afford a common activation mechanism in the NADPH oxidase system. The EPR results for cytochrome b558 activated with arachidonic acid showed that the transient high-spin ferric heme can bind cyanide. However, the high-spin ferric heme does not contribute to the O2*− production of cytochrome b558 in cell- free assays in the presence of cyanide.