Kinetic studies of the reduction of neutrophil cytochrome b-558 by dithionite.
The reduction with dithionite of neutrophil cytochrome b-558, implicated in superoxide generation by activated neutrophils, was investigated by a stopped-flow technique in non-ionic-detergent extracts of the membranes and in crude membrane particles. The dependence of the pseudo-first-order rate constants on the concentration of dithionite was consistent with a mechanism of reduction that involves the dithionite anion monomer SO2.- as the reactive species. The estimated second-order rate constant was 7.8 X 10(6) M-1 X S-1 for Lubrol PX-solubilized cytochrome b-558 and 5.1 X 10(6) M-1 X S-1 for the membrane-bound protein. The similarity of the kinetic constants suggests that solubilization did not introduce gross changes in the reactive site. Imidazole and p-chloromercuribenzoate, known as inhibitors of NADPH oxidase, did not affect significantly cytochrome b-558 reduction rates. The reaction rate of cytochrome b-558 with dithionite exhibited a near-zero activation energy. The first-order rate constant for reduction decreased with increasing ionic strength, indicating a positive effective charge on the reacting protein.
D002729:Chloromercuribenzoates / Q000494:pharmacology; D003573:Cytochrome b Group / Q000097:blood*; D004227:Dithionite; D006420:Hemeproteins / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013447:Sulfites; D013481:Superoxides / Q000097:blood; D013696:Temperature; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D003573:Cytochrome b Group; D006420:Hemeproteins; D007093:Imidazoles; D013447:Sulfites; D013481:Superoxides; D004227:Dithionite; D020245:p-Chloromercuribenzoic Acid; C029899:imidazole; C036888:cytochrome b558; D019255:NADPH Oxidases