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[Fe]‐hydrogenase harbors the iron‐guanylylpyridinol (FeGP) cofactor, in which the Fe(II) complex contains acyl‐carbon, pyridinol‐nitrogen, cysteine‐thiolate and two CO as ligands. Irradiation with UV‐A/blue light decomposes the FeGP cofactor to a 6‐carboxymethyl‐4‐guanylyl‐2‐pyridone (GP) and other components. Previous in vitro biosynthesis experiments indicated that the acyl‐ and CO‐ligands in the FeGP cofactor can scramble, but whether scrambling occurred during biosynthesis or photolysis was unclear. Here, we demonstrate that the [18O1‐carboxy]‐group of GP is incorporated into the FeGP cofactor by in vitro biosynthesis. MS/MS analysis of the 18O‐labeled FeGP cofactor revealed that the produced [18O1]‐acyl group is not exchanged with a CO ligand of the cofactor, indicating that the acyl and CO ligands are scrambled during photolysis rather than biosynthesis, which ruled out any biosynthesis mechanisms allowing acyl/CO ligands scrambling. Time‐resolved infrared spectroscopy indicated that an acyl‐Fe(CO)3 intermediate is formed during photolysis, in which scrambling of the CO and acyl ligands can occur. This finding also suggests that the light‐excited FeGP cofactor has a higher affinity for external CO. These results contribute to our understanding of the biosynthesis and photosensitive properties of this unique H2‐activating natural complex. [ABSTRACT FROM AUTHOR]

A de novo designed class of peptide‐based fluoropolymers composed of fluorinated aliphatic amino acids as main components is reported. Structural characterization provided insights into fluorine‐induced alterations on β‐strand to α‐helix transition upon an increase in SDS content and revealed the unique formation of PPII structures for trifluorinated fluoropeptides. A combination of circular dichroism, fluorescence‐based leaking assays and surface enhanced infrared absorption spectroscopy served to examine the insertion and folding processes into unilamellar vesicles. While partitioning into lipid bilayers, the degree of fluorination conducts a decrease in α‐helical content. Furthermore, this study comprises a report on the proteolytic stability of peptides exclusively built up by fluorinated amino acids and proved all sequences to be enzymatically degradable despite the degree of fluorination. Herein presented fluoropeptides as well as the distinctive properties of these artificial and polyfluorinated foldamers with enzyme‐degradable features will play a crucial role in the future development of fluorinated peptide‐based biomaterials. [ABSTRACT FROM AUTHOR]

The reconstitution of [Mn]‐hydrogenases using a series of MnI complexes is described. These complexes are designed to have an internal base or pro‐base that may participate in metal–ligand cooperative catalysis or have no internal base or pro‐base. Only MnI complexes with an internal base or pro‐base are active for H2 activation; only [Mn]‐hydrogenases incorporating such complexes are active for hydrogenase reactions. These results confirm the essential role of metal–ligand cooperation for H2 activation by the MnI complexes alone and by [Mn]‐hydrogenases. Owing to the nature and position of the internal base or pro‐base, the mode of metal–ligand cooperation in two active [Mn]‐hydrogenases is different from that of the native [Fe]‐hydrogenase. One [Mn]‐hydrogenase has the highest specific activity of semi‐synthetic [Mn]‐ and [Fe]‐hydrogenases. This work demonstrates reconstitution of active artificial hydrogenases using synthetic complexes differing greatly from the native active site. [ABSTRACT FROM AUTHOR]

[Fe]‐hydrogenase (Hmd) catalyzes the reversible hydrogenation of methenyl‐tetrahydromethanopterin (methenyl‐H4MPT+) with H2. H4MPT is a C1‐carrier of methanogenic archaea. One bacterial genus, Desulfurobacterium, contains putative genes for the Hmd paralog, termed HmdII, and the HcgA–G proteins. The latter are required for the biosynthesis of the prosthetic group of Hmd, the iron–guanylylpyridinol (FeGP) cofactor. This finding is intriguing because Hmd and HmdII strictly use H4MPT derivatives that are absent in most bacteria. We identified the presence of the FeGP cofactor in D. thermolithotrophum. The bacterial HmdII reconstituted with the FeGP cofactor catalyzed the hydrogenation of derivatives of tetrahydrofolate, the bacterial C1‐carrier, albeit with low enzymatic activities. The crystal structures show how Hmd recognizes tetrahydrofolate derivatives. These findings have an impact on future biotechnology by identifying a bacterial Hmd paralog. [Fe]‐hydrogenase (Hmd) catalyzes the hydrogenation of methenyl‐tetrahydromethanopterin (methenyl‐H4MPT+). H4MPT is a C1‐carrier of methanogenic archaea. One bacterial genus contains the Hmd paralog, HmdII. Bacterial HmdII catalyzes the hydrogenation of derivatives of the bacterial C1‐carrier, tetrahydrofolate. The crystal structure of HmdII explains the evolutionary adaptation to the different hydride‐accepting substrate. [ABSTRACT FROM AUTHOR]

Mono‐iron hydrogenase ([Fe]‐hydrogenase) reversibly catalyzes the transfer of a hydride ion from H2 to methenyltetrahydromethanopterin (methenyl‐H4MPT+) to form methylene‐H4MPT. Its iron guanylylpyridinol (FeGP) cofactor plays a key role in H2 activation. Evidence is presented for O2 sensitivity of [Fe]‐hydrogenase under turnover conditions in the presence of reducing substrates, methylene‐H4MPT or methenyl‐H4MPT+/H2. Only then, H2O2 is generated, which decomposes the FeGP cofactor; as demonstrated by spectroscopic analyses and the crystal structure of the deactivated enzyme. O2 reduction to H2O2 requires a reductant, which can be a catalytic intermediate transiently formed during the [Fe]‐hydrogenase reaction. The most probable candidate is an iron hydride species; its presence has already been predicted by theoretical studies of the catalytic reaction. The findings support predictions because the same type of reduction reaction is described for ruthenium hydride complexes that hydrogenate polar compounds. [ABSTRACT FROM AUTHOR]

[Fe]-hydrogenase (Hmd), an enzyme of the methanogenic energy metabolism, harbors an iron-guanylylpyridinol (Fe GP) cofactor used for H2 cleavage. The generated hydride is transferred to methenyl-tetrahydromethanopterin (methenyl-H4 MPT+). Most hydrogenotrophic methanogens contain the hmd-related genes hmd II and hmd III. Their function is still elusive. We were able to reconstitute the Hmd II holoenzyme of Methanocaldococcus jannaschii with recombinantly produced apoenzyme and the Fe GP cofactor, which is a prerequisite for in vitro functional analysis. Infrared spectroscopic and X-ray structural data clearly indicated binding of the Fe GP cofactor. Methylene-H4 MPT binding was detectable in the significantly altered infrared spectra of the Hmd II holoenzyme and in the Hmd II apoenzyme-methylene-H4 MPT complex structure. The related binding mode of the Fe GP cofactor and methenyl-H4 MPT+ compared with Hmd and their multiple contacts to the polypeptide highly suggest a biological role in Hmd II. However, holo-Hmd II 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 Hmd II 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 Hmd II as Hmd isoenzyme, H2 sensor, Fe GP-cofactor storage protein and scaffold protein for Fe GP-cofactor biosynthesis could be excluded. Inspired by the recently found binding of Hmd II to aminoacyl- tRNA synthetases and tRNA, we tentatively consider Hmd II as a regulatory protein for protein synthesis that senses the intracellular methylene-H4 MPT concentration. Database Structural data are available in the Protein Data Bank under the accession numbers ; ; and . [ABSTRACT FROM AUTHOR]

Abstract: [Fe]-Hydrogenase catalyzes the reversible activation of H2. CO and CN− inhibit this enzyme with low affinity (K i ≅0.1mM) by binding to the iron site of the bound iron-guanyrylpyridinol cofactor. We report here that isocyanides, which are formally isoelectronic with CO and CN−, strongly inhibit [Fe]-hydrogenase (K i as low as 1nM). The [NiFe]- and [FeFe]-hydrogenases tested were not inhibited by isocyanides. UV–Vis and infrared spectra revealed that the isocyanides bind to the iron center of [Fe]-hydrogenase. The inhibition kinetics are in agreement with the proposed catalytic mechanism, including the open/closed conformational change of the enzyme. [ABSTRACT FROM AUTHOR]

Abstract: [Fe]-hydrogenase is one of three types of enzymes known to activate H2. Crystal structure analysis recently revealed that its active site iron is ligated square-pyramidally by Cys176-sulfur, two CO, an “unknown” ligand and the sp2-hybridized nitrogen of a unique iron–guanylylpyridinol-cofactor. We report here on the structure of the C176A mutated enzyme crystallized in the presence of dithiothreitol (DTT). It suggests an iron center octahedrally coordinated by one DTT-sulfur and one DTT-oxygen, two CO, the 2-pyridinol’s nitrogen and the 2-pyridinol’s 6-formylmethyl group in an acyl-iron ligation. This result led to a re-interpretation of the iron ligation in the wild-type. [Copyright &y& Elsevier]