Crystal Structures of [Fe]-Hydrogenase from Methanolacinia paynteri Suggest a Path of the FeGP-Cofactor Incorporation Process

Abstract

[Fe]-hydrogenase (Hmd) catalyzes the reversible heterolytic cleavage of H2, and hydride transfer to methenyl-tetrahydromethanopterin (methenyl-H4MPT+). The iron-guanylylpyridinol (FeGP) cofactor, the prosthetic group of Hmd, can be extracted from the holoenzyme and inserted back into the protein. Here, we report the crystal structure of an asymmetric homodimer of Hmd from Methanolacinia paynteri (pHmd), which was composed of one monomer in the open conformation with the FeGP cofactor (holo-form) and a second monomer in the closed conformation without the cofactor (apo-form). In addition, we report the symmetric pHmd-homodimer structure in complex with guanosine monophosphate (GMP) or guanylylpyridinol (GP), in which each ligand was bound to the protein, where the GMP moiety of the FeGP-cofactor is bound in the holo-form. Binding of GMP and GP modified the local protein structure but did not induce the open conformation. The amino-group of the Lys150 appears to interact with the 2-hydroxy group of pyridinol ring in the pHmd–GP complex, which is not the case in the structure of the pHmd–FeGP complex. Lys150Ala mutation decreased the reconstitution rate of the active enzyme with the FeGP cofactor at the physiological pH. These results suggest that Lys150 might be involved in the FeGP-cofactor incorporation into the Hmd protein in vivo.

Acyl and CO Ligands in the [Fe]‐Hydrogenase Cofactor Scramble upon Photolysis

“…By mixing the extracted FeGP cofactor with the [Fe]‐hydrogenase apoenzyme, the FeGP cofactor binds to the protein and the fully active [Fe]‐hydrogenase holoenzyme is constituted. Upon generation of the holoenzyme, the external ligands are exchanged with a cysteine‐thiolate of the protein and a water molecule [3] . Accordingly, semisynthetic [Fe]‐hydrogenase was constructed by mixing mimic complexes and the apoenzyme [7]…”

Acyl and CO Ligands in the [Fe]‐Hydrogenase Cofactor Scramble upon Photolysis

“…[Fe]‐hydrogenase contains the iron‐guanylylpyridinol (FeGP) cofactor as the prosthetic group, in which a mono‐nuclear low spin Fe(II) is complexed with the 6‐acylmethyl substituent of the pyridinol, the pyridinol nitrogen, two CO ligands, and a cysteine thiolate (Figure 1a). [2] The cysteine‐thiolate anchors the cofactor to the protein [2d,3] . CO ligands are also observed in the dinuclear metal complexes of [NiFe]‐ and [FeFe]‐hydrogenases; [4] however, the acyl ligand is unique to [Fe]‐hydrogenase.…”

“…Upon generation of the holoenzyme, the external ligands are exchanged with a cysteinethiolate of the protein and a water molecule. [3] Accordingly, semisynthetic [Fe]-hydrogenase was constructed by mixing mimic complexes and the apoenzyme. [7] Irradiation with UV-A/blue light decomposes the FeGP cofactor in both the enzyme-bound and extracted forms.…”