ELECTRON-TRANSFER ACROSS THE O-2(-) GENERATING FLAVOCYTOCHROME-B OF NEUTROPHILS - EVIDENCE FOR A TRANSITION FROM A LOW-SPIN STATE TO A HIGH-SPIN STATE OF THE HEME IRON COMPONENT

The NADPH oxidase complex of activated neutrophils consists of a membr ane-bound flavocytochrome b and cytosolic activation factors. Despite its ability to react with Oz, the heme b component of the flavocytochr ome is insensitive to cyanide and CO, and slowly reactive to butyl iso cyanide, We report here that arachidonic acid, an anionic amphophil wh ich elicits oxidase activation in a cell-free system induces a transit ion of the heme iron of the neutrophil flavocytochrome b from a low-sp in hexacoordinated state to a high-spin pentacoordinated state and pro motes the binding of butyl isocyanide to the heme b. Low-temperature E PR spectra of air-oxidized flavocytochrome b either purified or in its membrane-bound form showed a low-spin signal at g = 3.26 and a high-s pin signal at g = 6.0. Upon addition of arachidonic acid, the g = 3.26 signal vanished; a low-spin signal at a = 2.23 appeared, and the sign al at g = 6.0 progressively increased, The subsequent addition of buty l isocyanide resulted in the decrease of the g = 6.0 and g = 2.23 sign als and in the appearance of a new low-spin signal at g = 2.33. Consis tent with the EPR results, upon addition of arachidonic acid to oxidiz ed flavocytochrome b, a 2.5 nm blue shift of the Sent peak was detecte d in low-temperature optical spectra, The subsequent addition of butyl isocyanide resulted in the emergence of a peak at 432 nm reflecting t he formation of a butyl isocyanide-oxidized heme b complex. In the cas e of sodium dithionite-reduced flavocytochrome b, arachidonic acid pro moted the binding of butyl isocyanide to the reduced heme b, as shown by the emergence of a peak at 434 nm and the decrease of the a band at 558 nm, The same promoting effect was encountered with sodium dodecyl sulfate, an anionic amphophil capable of eliciting oxidase activation like arachidonic acid. In contrast to arachidonic acid, arachidonic a cid methyl ester was ineffective and counteracted the effect of arachi donic acid. Butyl isocyanide added to intact neutrophils was found to bind to heme b, only after the cells have been activated. These data d emonstrate the transient accumulation of a pentacoordinated form of th e heme iron of flavocytochrome b under in vitro and in vivo conditions ; the pentacoordinated form of the reduced heme b is postulated to rea ct with O-2 to generate the superoxide anion.