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Iron–sulfur (Fe–S) centers are versatile building blocks in biological electron transfer chains because their redox potentials may cover a wide potential range depending on the type of the cluster and the specific protein environment. Resonance Raman (RR) spectroscopy is widely used to analyze structural properties of such cofactors, but it remains still a challenge to disentangle the overlapping signals of metalloproteins carrying several Fe–S centers. In this work, we combined RR spectroscopy with protein engineering and X‐ray crystallography to address this issue on the basis of the oxygen‐tolerant membrane‐bound hydrogenase from Ralstonia eutropha that catalyzes the reversible conversion of hydrogen into protons and electrons. Besides the NiFe‐active site, this enzyme harbors three different Fe–S clusters constituting an electron relay with a distal [4Fe–4S], a medial [3Fe–4S], and an unusual proximal [4Fe–3S] cluster that may carry a hydroxyl ligand in the superoxidized state. RR spectra were measured from protein crystals by varying the crystal orientation with respect to the electric field vector of the incident laser to achieve a preferential RR enhancement for individual Fe–S clusters. In addition to spectral discrimination by selective reduction of the proximal cluster, protein engineering allowed for transforming the proximal and medial cluster into standard cubane‐type [4Fe–4S] centers in the C19G/C120G and P242C variants, respectively. The latter variant was structurally characterized for the first time in this work. Altogether, the entirety of the RR data provided the basis for identifying the vibrational modes characteristic of the various cluster states in this "model" enzyme as a prerequisite for future studies of complex (FeS)‐based electron transfer chains. [ABSTRACT FROM AUTHOR]

Molecularly imprinted polymers (MIPs) are artificial receptors equipped with selective recognition sites for target molecules. One of the most promising-strategies for protein MIPs relies on the exploitation of short surface-exposed protein fragments, termed epitopes, as templates to imprint binding sites in a polymer scaffold for a desired protein. However, the lack of high-resolution structural data of flexible surface-exposed regions challenges the selection of suitable epitopes. Here, we addressed this drawback by developing a polyscopoletin-based MIP that recognizes recombinant proteins via the widely used Strep-tag II affinity peptide. Electrochemistry, surface-sensitive spectroscopy, and molecular dynamics simulations were employed to ensure an utmost control of the Strep-MIP electrosynthesis. The functionality of this novel platform was verified with two Strep-tag labeled enzymes: an O2-tolerant [NiFe]-hydrogenase, and an alkaline phosphatase. The enzymes preserved their biocatalytic activities after multiple utilization confirming the efficiency of Strep-MIP as a general biocompatible platform to confine recombinant proteins for exploitation in biotechnology., (© 2024 Wiley‐VCH GmbH.)

The membrane-bound [NiFe]-hydrogenase of Cupriavidus necator is a rare example of a truly O 2 -tolerant hydrogenase. It catalyzes the oxidation of H 2 into 2e - and 2H + in the presence of high O 2 concentrations. This characteristic trait is intimately linked to the unique Cys 6 [4Fe-3S] cluster located in the proximal position to the catalytic center and coordinated by six cysteine residues. Two of these cysteines play an essential role in redox-dependent cluster plasticity, which bestows the cofactor with the capacity to mediate two redox transitions at physiological potentials. Here, we investigated the individual roles of the two additional cysteines by replacing them individually as well as simultaneously with glycine. The crystal structures of the corresponding MBH variants revealed the presence of Cys 5 [4Fe-4S] or Cys 4 [4Fe-4S] clusters of different architecture. The protein X-ray crystallography results were correlated with accompanying biochemical, spectroscopic and electrochemical data. The exchanges resulted in a diminished O 2 tolerance of all MBH variants, which was attributed to the fact that the modified proximal clusters mediated only one redox transition. The previously proposed O 2 protection mechanism that detoxifies O 2 to H 2 O using four protons and four electrons supplied by the cofactor infrastructure, is extended by our results, which suggest efficient shutdown of enzyme function by formation of a hydroxy ligand in the active site that protects the enzyme from O 2 binding under electron-deficient conditions., Competing Interests: The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to P. S. (patrick.scheerer@charite.de), O. L. (oliver.lenz@tu-berlin.de), and I. Z. (ingo.zebger@tu-berlin.de)., (This journal is © The Royal Society of Chemistry.)