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Insights into the Nitric Oxide Reductase Mechanism of Flavodiiron Proteins from a Flavin-Free Enzyme.
- Source :
-
Biochemistry . 8/24/2010, Vol. 49 Issue 33, p7040-7049. 10p. - Publication Year :
- 2010
-
Abstract
- Flavodiiron proteins (FDPs) catalyze reductive scavenging of dioxygen and nitric oxide in air- sensitive microorganisms. FDPs contain a distinctive non-heme diiron/flavin mononucleotide (FMN) active Site. Alternative mechanisms for the nitric oxide reductase (NOR) activity consisting of either protonation ofa diiron-hridging hyponitrite or "super-reduction" of a diferrous-dinitrosyl by the proximal FMNH2 in the rate-determining step have been proposed. To test these alternative mechanisms, we examined a deflavinated FDP (deflavo-FDP) from Thermotoga maritima. The deflavo-FDP retains an intact diiron Site but does not exhibit multiturnover NOR or O2 reductase (O2R) activity. Reactions of the reduced (diferrous) deulavo-FDP with nitric oxide were examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. Anaerobic addition of nitric oxide up to one NO per diferrous deflavo-FDP results in formation of a diiron-mononitrosyl complex characterized by a broad S = ⅛ EPR signal arising from antiferromagnetic coupling of an S = ⅜ (FeNO)7 with an S = 2 Fe(II). Further addition of NO results in two reaction pathways, one of which produces N2O and the diferric site and the other of which produces a stable diiron-dinitrosyl complex. Both NO-treated and as-isolated deflavo-FDPs regain full NOR and O2R activities upon simple addition of FMN. The production of N2O upon addition of NO to the mononitrosyl deflavo-FDP supports the hyponitrite mechanism, but the concomitant formation of a stable diiron-dinitrosyl complex in the deflavo- FDP is consistent with a super-reduction pathway in the flavinated enzyme. We conclude that a diiron- mononitrosyl complex is an intermediate in the NOR catalytic cycle of FDPs. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 00062960
- Volume :
- 49
- Issue :
- 33
- Database :
- Academic Search Index
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 53450608
- Full Text :
- https://doi.org/10.1021/bi100788y