X-ray Crystal Structures ofMoorella thermoaceticaFprA. Novel Diiron Site Structure and Mechanistic Insights into a Scavenging Nitric Oxide Reductase^,

Abstract

Several members of a widespread class of bacterial and archaeal metalloflavoproteins, called FprA, likely function as scavenging nitric oxide reductases (S-NORs). However, the only published X-ray crystal structure of an FprA is for a protein characterized as a rubredoxin:dioxygen oxidoreductase (ROO) from Desulfovibrio gigas. Therefore, the crystal structure of Moorella thermoacetica FprA, which has been established to function as an S-NOR, was solved in three different states: as isolated, reduced, and reduced, NO-reacted. As is the case for D. gigas ROO, the M. thermoacetica FprA contains a solvent-bridged non-heme, non-sulfur diiron site with five-coordinate iron centers bridged by an aspartate, and terminal glutamate, aspartate, and histidine ligands. However, the M. thermoacetica FprA diiron site showed four His ligands, two to each iron, in all three states, whereas the D. gigas ROO diiron site was reported to contain only three His ligands, even though the fourth His residue is conserved. The Fe1-Fe2 distance within the diiron site of M. thermoacetica FprA remained at 3.2-3.4 A with little or no movement of the protein ligands in the three different states and with conservation of the two proximal open coordination sites. Molecular modeling indicated that each open coordination site can accommodate an end-on NO. This relatively rigid and symmetrical diiron site structure is consistent with formation of a diferrous dinitrosyl as the committed catalytic intermediate leading to formation of N(2)O. These results provide new insight into the structural features that fine-tune biological non-heme diiron sites for dioxygen activation vs nitric oxide reduction.

Spectroscopic characterization of heme iron–nitrosyl species and their role in NO reductase mechanisms in diiron proteins

“…90,156,157 A recombinant form of flavoprotein A from Moorella thermoacetica, which exhibits comparable NO reductase activity to denitrifying NORs, was recently crystallized. 87 In this protein, the active site contains a non-heme diiron cluster with two histidines per iron and two bridging ligands: a bidentate carboxylate group and a solvent molecule. 87 Carboxylate groups have been shown to provide structural flexibility at the active sites of non-heme diiron proteins (for an extreme example, see recent crystal structures of nigerythrin, which reveal a 2 Å movement of one iron associated with a Glu ↔ His ligand "toggling" at one iron 158 ).…”

Operon flv4-flv2 Provides Cyanobacterial Photosystem II with Flexibility of Electron Transfer

“…Surface-bound metals were found also in homologous structures. For example, the flavodoxin-like domain from Synechococcus sp (PDB ID: 3HLY) has a Ca 2+ , and M. thermoacetica FprA (PDB ID: 1YCF, 1YCG, and 1YCH) has several Zn 2+ bound at the surface (Silaghi-Dumitrescu et al, 2005). The metal ions on the structures originate from the crystallization solution, but some of the metal binding residues are conserved or substituted with similar residues in our model (see Supplemental Table 2 online).…”

“…FDPs form a complex group of enzymes, and many of them have been thoroughly characterized. Purified FDPs have been shown to function as homodimers or homotetramers (Vicente et al, 2008b(Vicente et al, , 2009, and many structures have been solved by x-ray crystallography (Frazão et al, 2000;Silaghi-Dumitrescu et al, 2005;Seedorf et al, 2007;Di Matteo et al, 2008). All members of the FDP family share a common minimal core containing two structural domains.…”

Flavodiiron Oxygen Reductase from Entamoeba histolytica

“…1): an N-terminal metallo-␤-lactamase-like domain harboring the catalytic diiron site, followed by a short chain flavodoxin-like domain containing a flavin mononucleotide (FMN) (2,4,10,(22)(23)(24). The three-dimensional structures revealed also that a "head to tail" dimeric arrangement is a pre-requisite for enzymatic function, allowing a close proximity between the FMN and the diiron center from opposing monomers, whereas within each monomer the minimal distance between the two cofactors is about 30 Å, precluding efficient electron transfer.…”