Spectroscopy and DFT Calculations of Flavo–Diiron Nitric Oxide Reductase Identify Bridging Structures of NO-Coordinated Diiron Intermediates

Abstract

Flavo-diiron proteins (FDPs) are widespread in anaerobic bacteria, archaea, and protozoa, where they serve as the terminal components of dioxygen and nitric oxide reductive scavenging pathways. FDPs contain an N,O-ligated diiron site adjacent to a flavin mononucleotide (FMN) cofactor. The diiron site is structurally similar to those in hemerythrin, ribonucleotide reductase, and methane monooxygenase. However, only FDPs turn over NO to N2O at significant rates and yields. Previous studies revealed sequential binding of two NO molecules to the diferrous site, forming mono- and dinitrosyl intermediates leading to N2O formation. In the present work, these mono- and dinitrosyl intermediates have been characterized by EPR and Mössbauer spectroscopies and DFT calculations. Our results show that the iron proximal to the cofactor binds the first NO to form the diiron mononitrosyl complex, implying the iron distal to the FMN binds the second NO to form the diiron dinitrosyl intermediate. The exchange-coupling constants, J (H = JS1·S2), were found to differ substantially, +17 cm−1 for the diiron mononitrosyl and +60 cm−1 for the diiron dinitrosyl. Notwithstanding this large difference, our findings indicate retention of at least one hydroxo bridge throughout the NOR catalytic cycle. The Mossbauer hyperfine parameters and DFT calculations confirmed a semibridging NO− ligand in the mononitrosyl intermediate that lowers the exchange parameter. The DFT calculations on the dinitrosyl intermediate suggest a contribution to J from direct exchange between the S = 1 spins on the NO− ligands, which could initiate N-N bond formation. Our results provide insight into why FDPs are the only known nonheme diiron enzymes that competently turn over NO to N2O.

A Nonheme Mononuclear {FeNO}7 Complex that Produces N2O in the Absence of an Exogenous Reductant

“…DFT calculations (vide infra) yield theoretical values of δ =0.44 mm s −1 , |Δ E Q |=0.99 mm s −1 for the {FeNO} 7 complex 2 , in reasonable agreement with experiment. The isomer shift for complex 2 is lower than that observed for the {FeNO} 7 unit in the deflavo‐(FDP)NO ( δ =0.68 mm s −1 ) suggesting a stronger polarization of the Fe−NO center towards Fe III −NO − [23] …”

The Biologically Relevant Coordination Chemistry of Iron and Nitric Oxide: Electronic Structure and Reactivity

“…This interaction leads to an increase in electron density in the NO­(π*) orbitals, resulting in the shift of the N–O stretch to lower energy. These ideas are further supported by DFT calculations . It is worth noting that DFT calculations further predict that in the absence of the semibridging binding mode for the hs-Fe II /hs-{FeNO} 7 intermediate, the exchange coupling constant J would increase by nearly a factor of 1.4, relative to that of the diferrous state .…”

“…When titrated with 1 equiv of NO per diiron site, this species forms at relatively high yield. It exhibits an S t = 1/2 EPR signal at g = 2.04 (for Tm FDP), which can be attributed to a hs-{FeNO} 7 ( S = 3/2) center AF coupled to a hs-Fe II ( S = 2) . Using temperature-dependent EPR data for the Tm FDP, the exchange coupling constant between the two iron sites was calculated to be J = −8.5 cm –1 .…”

Biological and Bioinspired Inorganic N–N Bond-Forming Reactions

“…Further, the same diFeNO pathway was implicated in the deflavinated form of the enzyme; the observation of the [{FeNO} 7 ] 2 and its competence for N 2 O formation without FMNH 2 is in disagreement with the sr pathway. 290 While Caranto and co-workers 286 Weitz and co-workers 292 further characterized the Fe 2+ {FeNO} 7 and [{FeNO} 7 ] 2 intermediates. Using a combination of EPR, 57 Fe Mössbauer and DFT methods, the authors conclude that the first NO binds to the proximal Fe (closest to the FMN cofactor), resulting in an exchange coupling constant of J = 17 cm −1 (Hex = JS 1 •S 2 ).…”