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Relevant academic research and scientific papers

Biosynthesis of the iron-guanylylpyridinol cofactor of [Fe]-hydrogenase in methanogenic archaea as elucidated by stable-isotope labeling

[Fe]-hydrogenase catalyzes the reversible hydride transfer from H 2 to methenyltetrahydromethanoptherin, which is an intermediate in methane formation from H2 and CO2 in methanogenic archaea. The enzyme harbors a unique active site iron-guanylylpyridinol (FeGP) cofactor, in which a low-spin FeII is coordinated by a pyridinol-N, an acyl group, two carbon monoxide, and the sulfur of the enzymes cysteine. Here, we studied the biosynthesis of the FeGP cofactor by following the incorporation of 13C and 2H from labeled precursors into the cofactor in growing methanogenic archaea and by subsequent NMR, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) and IR analysis of the isolated cofactor and reference compounds. The pyridinol moiety of the cofactor was found to be synthesized from three C-1 of acetate, two C-2 of acetate, two C-1 of pyruvate, one carbon from the methyl group of l-methionine, and one carbon directly from CO2. The metabolic origin of the two CO-ligands was CO2 rather than C-1 or C-2 of acetate or pyruvate excluding that the two CO are derived from dehydroglycine as has previously been shown for the CO-ligands in [FeFe]-hydrogenases. A formation of CO from CO2 via direct reduction catalyzed by a nickel-dependent CO dehydrogenase or from formate could also be excluded. When the cells were grown in the presence of 13CO, the two CO-ligands and the acyl group became 13C-labeled, indicating either that free CO is an intermediate in their synthesis or that free CO can exchange with these iron-bound ligands. Based on these findings, we propose pathways for how the FeGP cofactor might be synthesized.

Towards a functional identification of catalytically inactive [Fe]-hydrogenase paralogs

[Fe]-hydrogenase (Hmd), an enzyme of the methanogenic energy metabolism, harbors an iron-guanylylpyridinol (FeGP) cofactor used for H2 cleavage. The generated hydride is transferred to methenyl-tetrahydromethanopterin (methenyl-H4MPT+). Most hydrogenotrophic methanogens contain the hmd-related genes hmdII and hmdIII. Their function is still elusive. We were able to reconstitute the HmdII holoenzyme of Methanocaldococcus jannaschii with recombinantly produced apoenzyme and the FeGP cofactor, which is a prerequisite for in vitro functional analysis. Infrared spectroscopic and X-ray structural data clearly indicated binding of the FeGP cofactor. Methylene-H4MPT binding was detectable in the significantly altered infrared spectra of the HmdII holoenzyme and in the HmdII apoenzyme-methylene-H4MPT complex structure. The related binding mode of the FeGP cofactor and methenyl-H4MPT+ compared with Hmd and their multiple contacts to the polypeptide highly suggest a biological role in HmdII. However, holo-HmdII did not catalyze the Hmd reaction, not even in a single turnover process, as demonstrated by kinetic measurements. The found inactivity can be rationalized by an increased contact area between the C- and N-terminal folding units in HmdII compared with in Hmd, which impairs the catalytically necessary open-to-close transition, and by an exchange of a crucial histidine to a tyrosine. Mainly based on the presented data, a function of HmdII as Hmd isoenzyme, H2 sensor, FeGP-cofactor storage protein and scaffold protein for FeGP-cofactor biosynthesis could be excluded. Inspired by the recently found binding of HmdII to aminoacyl-tRNA synthetases and tRNA, we tentatively consider HmdII as a regulatory protein for protein synthesis that senses the intracellular methylene-H4MPT concentration. Database Structural data are available in the Protein Data Bank under the accession numbers 4YT8; 4YT2; 4YT4 and 4YT5. The genome of some methanogens contains besides the [Fe]-hydrogenase (Hmd) a related protein termed HmdII. We showed by X-ray and infrared spectroscopic data that HmdII binds iron-guanylylpyridinol, the Hmd specific cofactor, and methylene-tetrahydromethanopterin, the substrate of Hmd. However, the HmdII holoenzyme did not catalyze the Hmd reaction. We propose HmdII as a regulatory protein that senses the intracellular methylene-tetrahydromethanopterin concentration.