Kinetic characterization of the redox components in solubilized membranes from porcine neutrophils: reduction with dithionite and photoexcited NAD(P)H

Abstract

Cytochrome b558 in solubilized membranes prepared from porcine neutrophils was reduced by dithionite with a second-order rate constant of 2.5 x 10(6) M-1 s-1 at pH 7.4 and 20 degrees C accompanied by spectral changes with peaks at 428 nm and 560 nm and isosbestic points at 420 and 441 nm. When an anaerobic mixture of solubilized membranes and NAD(P)H was exposed to a white light, cytochrome b558 was reduced biphasically but with almost the same spectral profiles as in the dithionite reduction. Thus, participation of redox component(s) of unknown nature in the photochemical reduction was suggested. The NAD(P). radical generated by photoexcitation of NAD(P)H with a 355 nm laser pulse under anaerobic conditions also reduced cytochrome b558 with a high rate constant of 4.3 x 10(8) M-1 s-1 at pH 7.4 and 20 degrees C. The reduction of cytochrome b558 accompanied a simultaneous reduction of a component having an absorption band around 420 nm, suggesting participation of an iron-sulfur (Fe-S) cluster. The cytochrome b558 reduction was followed by its reoxidation by another component with an apparent second-order rate constant of 6.5 x 10(5) M-1 s-1. During the reoxidation, the Fe-S-like component remained in the reduced state, and thus its role other than as electron mediator in neutrophils NADPH oxidase is suggested. Not only the rate constant but also the extent of cytochrome b558 reoxidation decreased as the same reaction mixture was exposed to the laser pulse repeatedly. This result clearly indicates that an electron accumulates in this electron-accepting component designated tentatively as the omega component.