Abstract
Molecular properties of superoxide (O2-)-producing cytochrome b558 purified from neutrophils were investigated focusing on the mechanism of the catalytic reaction. The purified cytochrome, which was depleted of FAD, exhibited high O2(-)-generating activity with consumption of NADPH in the presence of microsomal NADPH-cytochrome P-450 reductase. Exogenous additions of CO, CN-, or N3- had no effect on the enzymatic activity. Potentiometric titration of the ferric-ferrous couple of the cytochrome showed that the midpoint reduction potential was -255 mV at pH 7.4. When the reaction of the reduced cytochrome with O2 was analyzed by stopped flow and rapid scanning spectrophotometry, the ferrous form was found to be converted to the ferric form at a rate constant of 9.3 x 10(6) M-1 s-1 at 10 degrees C without showing formation of an oxygenated intermediate. EPR measurement of the ferric cytochrome at 10 K showed that the electronic spin state was in a low spin with g values of 3.2, 2.05, and 1.5. These results suggest that the heme in a six-coordinated low spin state catalyzes one electron reduction of O2 without ligation of O2 to the heme iron during the catalytic cycle.
Highlights
From the Instituteof Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-01, §Molecular Biochemistry I, Faculty of Science, Himeji Institute of Technology, Akou-gun, Hyougo 678-12, and the YDepartmenotf Public Health, Faculty of Medicine, Kyoto University, Kyoto 606, Japan
It has long been postulated that cytochrome bsss functions astheterminal oxidizingenzyme of theNADPH oxidase tometry, the ferrous form was found to be converted based upon the kinetic and thermodynamic aspects derived to the ferric form at a rate constant of 9.3 X 10’ from studieswithneutrophilmembranes (13, 14)andits
EPR measurement of the ferric cytochrome at 10 K showed that the electronic spin state wasin a low spin with g values of 3.2, 2.05,and 1.5. These results suggest that the heme in a six-coordinated low spin state catalyzes one electron reduction of O2without ligation of O2to the heme iron during the catalytic cycle
Summary
Superoxide-generating Activity and Flavin Contents-Cytochrome b55s was purifiedfrom neutrophilmembranes by detergent solubilization andsubsequenthreechromatographic steps. The activity at a lower rate was observed by the use of theputidaredoxinreductasewith higher concentration of putidaredoxin (not shown) These results indicate that only NADPH-cytochrome P-450 reductase in tfhlaevoproteins has a specific site for interacting with cytochrome bsss and for mediating electrons to the cytochrome. During the purifica- the theoretical curve based on the Nernst equation for one tion, the specific content of the flavins decreased,whereas electron transition( n = 1)with E,,, of -255 mV (Fig. 3) This t h a t of the heme increased. The preparations after the gel agrees with the results on the titrationof the cytochrome in filtration contained essentially noflavins compared with the neutrophilmembranes (13, 14)and adetergent-solubilized heme content. These results indicate no involvemeonft flav- preparation (28). The reduced cytochrome was mixed with an oxyputidaredoxin reductase (-260 mV) with or without putida- genated solution in a stopped flow cell, andthespectral
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