How does a valine residue that modulates heme-NO binding kinetics in inducible NO synthase regulate enzyme catalysis?
Nitric oxide (NO) release from nitric oxide synthases (NOSs) depends on the dissociation of a ferric heme-NO product complex (Fe(III)NO) that forms immediately after NO is made in the heme pocket. The NOS-like enzyme of Bacillus subtilis (bsNOS) has 10-20 fold slower Fe(III)NO dissociation rate (k(d)) and NO association rate (k(on)) compared to mammalian NOS counterparts. We previously showed that an Ile for Val substitution at the opening of the heme pocket in bsNOS contributes to these differences. The complementary mutation in mouse inducible NOS oxygenase domain (Val346Ile) decreased the NO k(on) and k(d) by 8 and 3-fold, respectively, compared to wild-type iNOSoxy, and also slowed the reductive processing of the heme-O(2) catalytic intermediate. To investigate how these changes affect steady-state catalytic behaviors, we generated and characterized the V346I mutant of full-length inducible NOS (iNOS). The mutant exhibited a 4-5 fold lower NO synthesis activity, an apparent uncoupled NADPH consumption, and formation of a heme-NO complex during catalysis that was no longer sensitive to solution NO scavenging. We found that these altered catalytic behaviors were not due to changes in the heme reduction rate or in the stability of the enzyme heme-O(2) intermediate, but instead were due to the slower NO k(on) and k(d) and a slower oxidation rate of the enzyme ferrous heme-NO complex. Computer simulations that utilized the measured kinetic values confirmed this interpretation, and revealed that the V346I iNOS has an enhanced NADPH-dependent NO dioxygenase activity that converts almost 1 NO to nitrate for every NO that the enzyme releases into solution. Together, our results highlight the importance of heme pocket geometry in tuning the NO release versus NO dioxygenase activities of iNOS.
Wang
Zhi-Qiang
ZQ
Department of Chemistry, Kent State University Tuscarawas, New Philadelphia, OH 44663, United States. zwang3@kent.edu
Wei
Chin-Chuan
CC
Stuehr
Dennis J
DJ
D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology; D001665:Binding Sites; D002384:Catalysis; D003198:Computer Simulation; D006418:Heme / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D010084:Oxidation-Reduction; D013481:Superoxides / Q000378:metabolism; D014633:Valine / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D009566:Nitrates; D009573:Nitrites; D013481:Superoxides; D009569:Nitric Oxide; D006418:Heme; D009249:NADP; D052247:Nitric Oxide Synthase Type II; D014633:Valine