%0 Journal Article %T Spectroscopy and DFT Calculations of Flavo每Diiron Nitric Oxide Reductase Identify Bridging Structures of NO-Coordinated Diiron Intermediates %J - %D 2018 %R https://doi.org/10.1021/acscatal.8b03051 %X Flavo每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 Mˋssbauer 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每1 for the diiron mononitrosyl and +60 cm每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 Mˋssbauer hyperfine parameters and DFT calculations confirmed a semibridging NO每 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每 ligands, which could initiate N每N bond formation. Our results provide insight into why FDPs are the only known nonheme diiron enzymes that competently turn over NO to N2O %U https://pubs.acs.org/doi/10.1021/acscatal.8b03051