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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 [ 18 O 1 -carboxy]-group of GP is incorporated into the FeGP cofactor by in vitro biosynthesis. MS/MS analysis of the 18 O-labeled FeGP cofactor revealed that the produced [ 18 O 1 ]-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 H 2 -activating natural complex., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

The reconstitution of [Mn]-hydrogenases using a series of Mn I 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 Mn I complexes with an internal base or pro-base are active for H 2 activation; only [Mn]-hydrogenases incorporating such complexes are active for hydrogenase reactions. These results confirm the essential role of metal-ligand cooperation for H 2 activation by the Mn I 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., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

[Fe]-hydrogenase (Hmd) catalyzes the reversible hydrogenation of methenyl-tetrahydromethanopterin (methenyl-H 4 MPT + ) with H 2 . H 4 MPT 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 H 4 MPT 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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mono-iron hydrogenase ([Fe]-hydrogenase) reversibly catalyzes the transfer of a hydride ion from H 2 to methenyltetrahydromethanopterin (methenyl-H 4 MPT + ) to form methylene-H 4 MPT. Its iron guanylylpyridinol (FeGP) cofactor plays a key role in H 2 activation. Evidence is presented for O 2 sensitivity of [Fe]-hydrogenase under turnover conditions in the presence of reducing substrates, methylene-H 4 MPT or methenyl-H 4 MPT + /H 2 . Only then, H 2 O 2 is generated, which decomposes the FeGP cofactor; as demonstrated by spectroscopic analyses and the crystal structure of the deactivated enzyme. O 2 reduction to H 2 O 2 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., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

[Fe]-Hydrogenase (Hmd) catalyzes reversible hydride transfer from H 2 . It harbors an iron-guanylylpyridinol as a cofactor with an Fe II that is ligated to one thiolate, two COs, one acyl-C, one pyridinol-N, and solvent. Here, we report that Cu I and H 2 O 2 inactivate Hmd (half-maximal rates at 1 μM Cu I and 20 μM H 2 O 2 ) and that Fe II inhibits the enzyme with very high affinity (K i =40 nM). Infrared and EPR studies together with competitive inhibition studies with isocyanide indicated that Cu I exerts its inhibitory effect most probably by binding to the active site iron-thiolate ligand. Using the same methods, it was found that H 2 O 2 binds to the active-site iron at the solvent-binding site and oxidizes Fe II to Fe III . Also it was shown that Fe II reversibly binds away from the active site iron, with binding being competitive to the organic hydride acceptor; this inhibition is specific for Fe II and is reminiscent of that for the [FeFe]-hydrogenase second iron, which specifically interacts with H 2 ., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

New techniques in vibrational spectroscopy are promising for the study of biological samples as they provide exquisite spatial and/or temporal resolution with the benefit of minimal perturbation of the system during observation. In this Minireview we showcase the power of modern infrared techniques when applied to biological and biomimetic systems. Examples will be presented on how conformational changes in peptides can be traced with femtosecond resolution and nanometer sensitivity by 2D IR spectroscopy, and how surface-enhanced infrared difference absorption spectroscopy can be used to monitor the effect of the membrane potential on a single proton-transfer step in an integral membrane protein. Vibrational spectra of monolayers of molecules at basically any interface can be recorded with sum-frequency generation, which is strictly surface-sensitive. Chemical images are recorded by applying scanning near-field infrared microscopy at lateral resolutions better than 50 nm.