Computational Exploration of Enzyme Promiscuity: Mechanisms of O₂ and NO Reduction Activities of the Desulfovibrio gigas Flavodiiron Protein

Flavodiiron proteins possess reductive scavenging properties toward dioxygen and/or nitric oxide in various microorganisms. Among them, the Desulfovibrio gigas flavodiiron protein (Dg_ROO) was reported to be capable of catalyzing both O₂ and NO reduction reactions, though it displays higher activity in reducing O₂ than NO. In this study, quantum chemical methodology is employed to investigate the intricate mechanisms underlying these versatile reduction reactions catalyzed by Dg_ROO. The calculations demonstrated that the flavin mononucleotide (FMN) cofactor plays a pivotal role in the cleavage of O–O bonds during the four-electron reduction of O₂ by providing two protons and two electrons to the reaction site. The O–O bond could take place from two different metal oxidation states, namely, Fe^(II)Fe^(II)–HOOH or Fe^(II)Fe^(III)–OOH, depending on the rate for the electron/proton transfer from FMN to the diiron site. Subsequently, two water molecules are generated through two consecutive outer-sphere proton-coupled electron transfer steps. NO reduction is suggested to commence with the generation of a bridging hyponitrite (N–N) via the direct coupling of two NO molecules. A directional rotation of this hyponitrite species then yields an N–O bridging hyponitrite dianion, which triggers the cleavage of the N–O bond and generates N₂O. The electron/proton transfer from FMN to the diiron site transpires after forming the N₂O product. Further in-depth analyses and comparisons of these two reduction mechanisms highlight the crucial role played by two key second-shell tyrosine residues in the promiscuity and selectivity of Dg_ROO. These findings offer valuable insights into the functional diversity of other flavodiiron proteins (FDPs) and may contribute to a better understanding of their catalytic properties.