Transient kinetic studies of heme reduction in Escherichia coli nitrate reductase A (NarGHI) by menaquinol.
We have studied the transient kinetics of quinol-dependent heme reduction in Escherichia coli nitrate reductase A (NarGHI) by the menaquinol analogue menadiol using the stopped-flow method. Four kinetic phases are observed in the reduction of the hemes. A transient species, likely to be associated with a semiquinone radical anion, is observed with kinetics that correlates with one of the phases. The decay of the transient species and the formation of the second reduction phase of the hemes can be fitted to a double-exponential equation giving similar rate constants, k(1) = 9.24 +/- 0.9 s(-1) and k(2) = 0.22 +/- 0.02 s(-1) for the decay of the transient species, and k(1) = 9.23 +/- 0.9 s(-1) and k(2) = 0.22 +/- 0.02 s(-1) for the formation of the reduction phase. The quinol-binding-site inhibitors 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and stigmatellin have significant and different inhibitory effects on the reduction kinetics. The kinetics of heme reduction in NarI expressed in the absence of the NarGH catalytic dimer (NarI(DeltaGH) exhibits only two kinetic phases, and the decay of the transient species also correlates kinetically with the second reduction phase of the hemes. We have also studied nitrate-dependent heme reoxidation following quinol-dependent heme reduction using a sequential stopped-flow method. HOQNO elicits a much stronger inhibitory effect than stigmatellin on the reoxidation of the hemes. On the basis of our results, we propose schemes for the mechanism of NarGHI reduction by menaquinol and reoxidation by nitrate.
CIHR Membrane Protein Research Group, Department of Biochemistry, 474 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Zhongwei
Z
Zhao
Richard A
RA
Rothery
Joel H
JH
Weiner
D002384:Catalysis; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006912:Hydroxyquinolines / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009565:Nitrate Reductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009566:Nitrates / Q000494:pharmacology; D010084:Oxidation-Reduction; D011090:Polyenes / Q000494:pharmacology; D014812:Vitamin K / Q000031:analogs & derivatives* / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D006912:Hydroxyquinolines; D009566:Nitrates; D011090:Polyenes; D014812:Vitamin K; C001333:2-(n-heptyl)-4-hydroxyquinoline N-oxide; D006418:Heme; C041573:stigmatellin; D009565:Nitrate Reductases; C023885:menadiol