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 of a diiron-bridging 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) deflavo-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 = 1/2EPR signal arising from antiferromagnetic coupling of an S = 3/2 {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.
Original language | English (US) |
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Pages (from-to) | 7040-7049 |
Number of pages | 10 |
Journal | Biochemistry |
Volume | 49 |
Issue number | 33 |
DOIs | |
State | Published - Aug 24 2010 |
ASJC Scopus subject areas
- Biochemistry