The active form of the ferric heme in neutrophil cytochrome b(558) is low-spin in the reconstituted cell-free system in the presence of amphophil.
The spin state of the heme in superoxide (O(2)(.)(-))-producing cytochrome b(558) purified from pig neutrophils was examined by means of room-temperature magnetic circular dichroism (MCD) under physiological conditions. Cytochrome b(558) with varying amounts of low-spin and high-spin heme was prepared by either pH adjustment or heat treatment, and the O(2)(.)(-)-forming activity in a cell-free system was found to correlate with the low-spin heme content. The possibility that the O(2)(.)(-)-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 b(558) 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 b(558) 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 O(2)(.)(-) production of cytochrome b(558) in cell-free assays in the presence of cyanide.
Fujii
H
H
Department of Inflammation Research, The Tokyo Metropolitan Institute of Medical Science (Rinshoken), Bunkyo-ku, Tokyo, 113-8613, Japan. hfujii@shs.sapmed.ac.jp
Finnegan
M G
MG
Johnson
M K
MK
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D002474:Cell-Free System; D002942:Circular Dichroism; D003486:Cyanides / Q000494:pharmacology; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry*; D066298:In Vitro Techniques; D019814:Myristic Acid / Q000494:pharmacology; D019255:NADPH Oxidases; D009504:Neutrophils / Q000737:chemistry; D010084:Oxidation-Reduction; D013481:Superoxides / Q000378:metabolism; D013552:Swine
D003486:Cyanides; D003573:Cytochrome b Group; D019814:Myristic Acid; D013481:Superoxides; D016718:Arachidonic Acid; D006418:Heme; C036888:cytochrome b558; D019255:NADPH Oxidases