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Abstract

The di-iron flavoprotein F₄₂₀H₂ oxidase found in methanogenic Archaea catalyzes the four-electron reduction of O₂ to 2H₂O with 2 mol of reduced coenzyme F₄₂₀(7,8-dimethyl-8-hydroxy-5-deazariboflavin). We report here on crystal structures of the homotetrameric F₄₂₀H₂ oxidase from Methanothermobacter marburgensis at resolutions of 2.25 A, 2.25 A and 1.7 A, respectively, from which an active reduced state, an inactive oxidized state and an active oxidized state could be extracted. As found in structurally related A-type flavoproteins, the active site is formed at the dimer interface, where the di-iron center of one monomer is juxtaposed to FMN of the other. In the active reduced state [Fe(II)Fe(II)FMNH₂], the two irons are surrounded by four histidines, one aspartate, one glutamate and one bridging aspartate. The so-called switch loop is in a closed conformation, thus preventing F₄₂₀ binding. In the inactive oxidized state [Fe(III)FMN], the iron nearest to FMN has moved to two remote binding sites, and the switch loop is changed to an open conformation. In the active oxidized state [Fe(III)Fe(III)FMN], both irons are positioned as in the reduced state but the switch loop is found in the open conformation as in the inactive oxidized state. It is proposed that the redox-dependent conformational change of the switch loop ensures alternate complete four-electron O₂ reduction and redox center re-reduction. On the basis of the known Si-Si stereospecific hydride transfer, F₄₂₀H₂ was modeled into the solvent-accessible pocket in front of FMN. The inactive oxidized state might provide the molecular basis for enzyme inactivation by long-term O₂ exposure observed in some members of the FprA family.