Biochemical and spectroscopic insights into peculiar active site structures and biosynthesis of O2-tolerant [NiFe]-hydrogenases

[NiFe]-hydrogenases are metalloenzymes catalyzing the reversible cleavage of H2 into two H+ and two e–. They are particularly interesting for clean and sustainable H2-based energy conversion approaches or the H2-driven regeneration of nucleotide cofactors. Their active site consists of a NiFe(CN)2(CO) moiety coordinated to the protein by four strictly conserved cysteine residues. Two of them serve as terminal ligands to the Ni and two are bridging ligands coordinating both, the Ni and Fe. The Fe is additionally equipped with one carbon monoxide (CO) and two cyanide (CN–) ligands. The maturation of the NiFe(CN)2(CO) active site involves at least six auxiliary proteins, namely HypA-F. A seventh Hyp protein, HypX, is required for aerobic CO ligand biosynthesis. Although, the biosynthesis of [NiFe]-hydrogenases has been studied extensively, many details of their maturation still remain elusive. Because of its O2-tolerance and thermostability, the soluble, NAD+-reducing [NiFe]- hydrogenases from Hydrogenophilus thermoluteolus (HtSH) is of special biotechnological interest. Previous studies unveiled unusual structural and spectroscopic properties of the oxidized enzyme. Here, we present evidence by protein biochemistry in combination with infrared spectroscopy that the peculiar active site structure is causative for the unusual spectroscopic properties and probably related to a novel O2 protection mechanism. The second part of this thesis focused on the maturation of [NiFe]-hydrogenases, especially the assembly of the Fe(CN)2(CO) moiety. Therefore, purified HypCD was biochemically and spectroscopically investigated and apo-HypCD was used in in vitro maturation experiments. The preliminary results presented here, help to address open questions regarding the Fe(CN)2(CO) moiety assembly process, the order of ligand attachment as well as the coordination and characterization of the Fe(CN)2(CO) moiety and its transfer to the hydrogenase apo-large subunit. Furthermore, a comprehensive biochemical characterization of HypX revealed detailed insight into the mechanism of aerobic CO ligand synthesis. In the proposed mechanism, the formyl group of N10-formyl-THF is first transferred to Coenzyme A (CoA) producing formyl-CoA, which, in the second step, becomes decarbonylated to form CoA and CO.

Author dc:creator

• Schulz, Anne-Christine

Advisor dc:contributor.advisor

• Lenz, Oliver

Licence dc:rights.uri

• https://creativecommons.org/licenses/by/4.0/

Language dc:language.iso

en

Identifier URI

http://dx.doi.org/10.14279/depositonce-10960

OAI identifier oai:identifier

oai:depositonce.tu-berlin.de:11303/12085