a b s t r a c tThe HypC and HypD maturases are required for the biosynthesis of the Fe(CN) 2 CO cofactor in the large subunit of [NiFe]-hydrogenases. Using infrared spectroscopy we demonstrate that an anaerobically purified, Strep-tagged HypCD complex from Escherichia coli exhibits absorption bands characteristic of diatomic CO and CN À ligands as well as CO 2 . Metal and sulphide analyses revealed that along with the [4Fe-4S] 2+ cluster in HypD, the complex has two additional oxygen-labile Fe ions. We prove that HypD cysteine 41 is required for the coordination of all three ligands. These findings suggest that the HypCD complex carries minimally the Fe(CN) 2 CO cofactor.

During biosynthesis of [NiFe]-hydrogenase 2 (Hyd-2) of Escherichia coli, a 15-amino-acid C-terminal peptide is cleaved from the catalytic large subunit precursor, pro-HybC. This peptide is removed only after NiFe(CN) 2 CO cofactor insertion by the Hyp accessory protein machinery has been completed, suggesting that it has a regulatory function during enzyme maturation. We show here that in hyp mutants that fail to synthesize and insert the NiFe cofactor, and therefore retain the peptide, the Tat (twin-arginine translocon) signal peptide on the small subunit HybO is not removed and the subunit is degraded. In a mutant lacking the large subunit, the Tat signal peptide was also not removed from pre-HybO, indicating that the mature large subunit must actively engage the small subunit to elicit Tat transport. We validated the proposed regulatory role of the C-terminal peptide in controlling enzyme assembly by genetically removing it from the precursor of HybC, which allowed assembly and Tat-dependent membrane association of a HybC-HybO heterodimer lacking the NiFe(CN) 2 CO cofactor. Finally, genetic transfer of the C-terminal peptide from pro-HyaB, the large subunit of Hyd-1, onto HybC did not influence its dependence on the accessory protein HybG, a HypC paralog, or the specific protease HybD. This indicates that the C-terminal peptide per se is not required for interaction with the Hyp machinery but rather suggests a role of the peptide in maintaining a conformation of the protein suitable for cofactor insertion. Together, our results demonstrate that the C-terminal peptide on the catalytic subunit controls biosynthesis, assembly, and membrane association of Hyd-2. IMPORTANCE[NiFe]-hydrogenases are multisubunit enzymes with a catalytic subunit containing a NiFe(CN) 2 CO cofactor. Results of previous studies suggested that after synthesis and insertion of the cofactor by the Hyp accessory proteins, this large subunit changes conformation upon proteolytic removal of a short peptide from its C terminus. We show that removal of this peptide is necessary to allow the cleavage of the Tat signal peptide from the small subunit with concomitant membrane association of the heterodimer to occur. Genetic removal of the C-terminal peptide from the large subunit allowed productive interaction with the small subunit and Tat-dependent membrane insertion of a NiFe cofactor-free enzyme. Results based on swapping of C-terminal peptides between hydrogenases suggest that this peptide governs enzyme assembly via a conformational switch. Biosynthesis of complex metal cofactor-containing, multisubunit enzymes requires strict coordination of cofactor biosynthesis, subunit recruitment, and targeting of the protein to its final cellular location. This is particularly important for the numerous redox enzymes present in bacterial systems, many of which are membrane associated or indeed transported across the cytoplasmic membrane (1). Coordination of biosynthesis and assembly is important because cofactors are often highly complex and ma...

The denitrifying bacterium 'Aromatoleum aromaticum' strain EbN1 is one of the best characterized bacteria regarding anaerobic ethylbenzene degradation. EbN1 also degrades various other aromatic and phenolic compounds in the absence of oxygen, one of them being p-ethylphenol. Despite having similar chemical structures, ethylbenzene and p-ethylphenol have been proposed to be metabolized by completely separate pathways. In this study, we established and applied biochemical and molecular biological methods to show the (almost) exclusive presence and specificity of enzymes involved in the respective degradation pathways by recording enzyme activities, complemented by heme staining, immuno- and biotin-blotting analyses. These combined results substantiated the predicted p-ethylphenol degradation pathway. The identified enzymes include a heme c-containing p-ethylphenol-hydroxylase, both an (R)- and an (S)-specific alcohol dehydrogenase as well as a novel biotin-dependent carboxylase. We also establish an activity assay for benzoylacetate-CoA ligases likely being involved in both metabolic pathways.

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