Evidence for acyl–iron ligation in the active site of [Fe]-hydrogenase provided by mass spectrometry and infrared spectroscopy

Abstract

[Fe]-hydrogenase catalyzes the reversible heterolytic cleavage of H₂ and stereo-specific hydride transfer to the substrate methenyltetrahydromethanopterin in methanogenic archaea. This enzyme contains a unique iron guanylylpyridinol (FeGP) cofactor as a prosthetic group. It has recently been proposed—on the basis of crystal structural analyses of the [Fe]-hydrogenase holoenzyme—that the FeGP cofactor contains an acyl–iron ligation, the first one reported in a biological system. We report here that the cofactor can be reversibly extracted with acids; its exact mass has been determined by electrospray ionization Fourier transform ion cyclotron resonance mass-spectrometry. The measured mass of the intact cofactor and its gas-phase fragments are consistent with the proposed structure. The mass of the light decomposition products of the cofactor support the presence of acyl–iron ligation. Attenuated total reflection infrared spectroscopy of the FeGP cofactor revealed a band near wave number 1700 cm⁻¹, which was assigned to the CO (double bond) stretching mode of the acyl–iron ligand.