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

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Abstract

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.

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