Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 294, Issue 19, Pages 7904-7916Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA119.007810
Keywords
nitric oxide; nitric-oxide synthase; heme; enzyme mechanism; computational biology; thermodynamics; enzyme kinetics; dioxygenation; heme protein; heme-thiolate; reaction mechanism; redox reaction
Categories
Funding
- National Institutes of Health [GM51491]
- National Science Foundation [CHE-1464696]
- American Heart Association [0625632B]
Ask authors/readers for more resources
Nitric oxide (NO) synthases (NOSs) catalyze the formation of NO from l-arginine. We have shown previously that the NOS enzyme catalytic cycle involves a large number of reactions but can be characterized by a global model with three main rate-limiting steps. These are the rate of heme reduction by the flavin domain (k(r)), of dissociation of NO from the ferric heme-NO complex (k(d)), and of oxidation of the ferrous heme-NO complex (k(ox)). The reaction of oxygen with the ferrous heme-NO species is part of a futile cycle that does not directly contribute to NO synthesis but allows a population of inactive enzyme molecules to return to the catalytic cycle, and thus, enables a steady-state NO synthesis rate. Previously, we have reported that this reaction does involve the reaction of oxygen with the NO-bound ferrous heme complex, but the mechanistic details of the reaction, that could proceed via either an inner-sphere or an outer-sphere mechanism, remained unclear. Here, we present additional experiments with neuronal NOS (nNOS) and inducible NOS (iNOS) variants (nNOS W409F and iNOS K82A and V346I) and computational methods to study how changes in heme access and electronics affect the reaction. Our results support an inner-sphere mechanism and indicate that the particular heme-thiolate environment of the NOS enzymes can stabilize an N-bound Fe-III-N(O)OO- intermediate species and thereby catalyze this reaction, which otherwise is not observed or favorable in proteins like globins that contain a histidine-coordinated heme.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available