A Diferrous-Dinitrosyl Intermediate in the N₂O-Generating Pathway of a Deflavinated Flavo-Diiron Protein

Abstract

Flavo-diiron proteins (FDPs) function as anaerobic nitric oxide scavengers in some microorganisms, catalyzing reduction of nitric to nitrous oxide. The FDP from Thermotoga maritima can be prepared in a deflavinated form with an intact diferric site (deflavo-FDP). Hayashi et al. [(2010) Biochemistry 49, 7040–7049] reported that reaction of NO with reduced deflavo-FDP produced substoichiometric N2O. Here we report a multispectroscopic approach to identify the iron species in the reactions of deflavo-FDP with NO. Mössbauer spectroscopy identified two distinct ferrous species after reduction of the antiferromagnetically coupled diferric site. Approximately 60% of the total ferrous iron was assigned to a diferrous species associated with the N2O-generating pathway. This pathway proceeds through successive diferrous-mononitrosyl (S = 1/2 FeII{FeNO}7) and diferrous-dinitrosyl (S = 0 [{FeNO}7]2) species that form within ∼100 ms of mixing of the reduced protein with NO. The diferrous-dinitrosyl intermediate converted to an antiferromagnetically coupled diferric species that was spectroscopically indistinguishable from that in the starting deflavinated protein. These diiron species closely resembled those reported for the flavinated FDP [Caranto et al. (2014) J. Am. Chem. Soc. 136, 7981–7992], and the time scales of their formation and decay were consistent with the steady state turnover of the flavinated protein. The remaining ∼40% of ferrous iron was inactive in N2O generation but reversibly bound NO to give an S = 3/2 {FeNO}7 species. The results demonstrate that N2O formation in FDPs can occur via conversion of S = 0 [{FeNO}7]2 to a diferric form without participation of the flavin cofactor.

Dioxygen and nitric oxide scavenging by Treponema denticola flavodiiron protein: a mechanistic paradigm for catalysis

“…in Scheme 3) [37]. This rate-determining “spontaneous” decay is supported by the observation that deflavinated T. maritima diferrous FDP reacted with NO to form the same diferrous dinitrosyl species, which decayed to diferric with formation of N 2 O on the same time scale as the FMNH 2 oxidation in the flavinated FDP [38, 48]. In our proposed mechanism, the r.d.s.…”

“…The apparently higher affinity of the second NO for Td FDP red is opposite to that reported for T. maritima FDP red , for which the NOR reaction mechanism has recently been characterized in some detail [37, 48]. We, therefore, investigated whether these reversed NO relative affinities indicated a different NO reaction pathway for Td FDP.…”

“…This correlation is consistent with NOR turnover being rate-limited by a process that triggers two-electron FMNH 2 oxidation. Our detailed investigations of the NO reaction with T. maritima FDP red [37, 48] indicated that the rate-limiting process for NOR turnover is the “spontaneous” conversion of the diferrous dinitrosyl to diferric with release of N 2 O generating a transient FMNH 2 /Fe III Fe III species (Scheme 3). The proximal FMNH 2 then rapidly re-reduces the diferric site, resulting in FMN ox /Fe II Fe II .…”

Origin of Nitric Oxide Reduction Activity in Flavo–Diiron NO Reductase: Key Roles of the Second Coordination Sphere

“…Furthermore,t his viewpoint is in line with ap ure quantum mechanical (QM) theoretical study for another FDP. [15] In contrast, recent spectroscopic detection of [{FeNO} 7 ] 2 and its kinetic study in Tm FDP, supported N-N coupling between two bound NOs in [{FeNO} 7 ] 2 (Scheme 1a,i ), [16,17] also supported by the NO reduction reactivity of synthetic analogues of [{FeNO} 7 ] 2 . [18][19][20] Thes econd unclear mechanistic issue is the exact reduction timing of the flavin mononucleotide (FMN) cofactor, which can confer extra electrons to an earby diiron active center either after or during NO reduction to N 2 O( Scheme 1b).…”

“…[19b] Nevertheless,K urtz and Hendrich surprisingly showed that in Tm FDP,F MN is not necessary in the single turnover of NO reduction to N 2 O, but plays the non-catalytic role of recovering the diiron center from ad iferric to diferrous state after around of catalysis. [16,17] This mechanistic disparity concerning the timing of external reduction is still hard to understand.…”