Abstract
The active site of all [NiFe]-hydrogenases (Hyd) has a bimetallic NiFe(CN)2CO cofactor that requires the combined action of several maturation proteins for its biosynthesis and insertion into the precursor form of the large subunit of the enzyme. Cofactor insertion is an intricately controlled process, and the large subunit of almost all Hyd enzymes has a C-terminal oligopeptide extension that is endoproteolytically removed as the final maturation step. This extension might serve either as one of the recognition motifs for the endoprotease, as well as an interaction platform for the maturation proteins, or it could have a structural role to ensure the active site cavity remains open until the cofactor is inserted. To distinguish between these alternatives, we exchanged the complete C-terminal extension of the precursor of Escherichia coli hydrogenase 2 (Hyd-2) for the C-terminal extension of the Hyd-1 enzyme. Using in-gel activity staining, we demonstrate clearly that this large subunit precursor retains its specificity for the HybG maturation chaperone, as well as for the pro-HybC-specific endoprotease HybD, despite the C-terminal exchange. Bacterial two-hybrid studies confirmed interaction between HybD and the pro-HybC variant carrying the exchanged C-terminus. Limited proteolysis studies of purified precursor and mature HybC protein revealed that, in contrast to the precursor, the mature protein was protected against trypsin attack, signifying a major conformational change in the protein. Together, our results support a model whereby the function of the C-terminal extension during subunit maturation is structural.
Highlights
The wild-type strain MC4100 acted as a positive control in each experiment and revealed three sets of signals (Ballantine and Boxer, 1985; Pinske et al, 2011) representing H2-oxidizing enzyme activity, characteristic for the conditions used: Hyd-1 had the fastest mobility in the gel; hydrogenase 2 (Hyd-2) migrated as a double band, and occasionally a third band could be discerned; and near the origin of the gel a weakly staining activity band due to a secondary H2-oxidizing activity of the formate dehydrogenases FDH-O and FDH-N was observed (Soboh et al, 2011)
Exchange of amino acids AV for ST at amino acid positions 550-551, delivering pro-HybC§ST, resulted in an active Hyd-2 enzyme, albeit with a slightly weaker activity signal (Figure 2A). This result indicated that exchange of the AV for ST amino acid residues adjacent to the terminal Cys-549 residue only reduced the efficiency of maturation or processing of the large subunit precursor but did not prevent maturation of an active Hyd-2 enzyme
Our results support the conclusion that the C-terminal oligopeptide extension on the large subunit of Hyd-2 has a chaperone-like function (Theodoratou et al, 2005) and do not support the conclusion that it acts as a recognition motif for either the HybG or HybD hydrogenase maturation proteins
Summary
A still unresolved issue on the maturation pathway of the catalytic, or large, subunit of [NiFe]hydrogenases (Hyd) is what role the C-terminal oligopeptide extension plays during the maturation process: Does it act as a recognition motif allowing interaction with maturation proteins, or does it function as a form of “intra-molecular” chaperone that constrains the large subunit precursor in a conformation allowing access of the maturation machinery, which interacts elsewhere on the protein? Or does it do both?Conformational Constraint in Apo-HybCWith the exception of the large subunit of sensory and Ech hydrogenases (Theodoratou et al, 2005), the final step in the maturation process of the large subunit involves endoproteolytic cleavage of an approximately 15-amino acid-long oligopeptide from the C-terminus of the protein (Gollin et al, 1992; Menon et al, 1993; Rossmann et al, 1994; Böck et al, 2006). Nickel is the template recognized by the specific endoprotease (Theodoratou et al, 2000a) and cleavage occurs between the conserved His and Val residues, which are located three and four amino acid residues, respectively, C-terminal to the Cys-4 residue that coordinates the cofactor (Figure 1A). Proteolytic removal of the C-terminal oligopeptide causes a conformational switch in the complete protein bringing the Cys-4 residue (Figures 1A,C) into coordination distance to bridge both metals and “fix” the cofactor firmly within the active site (Magalon and Böck, 2000; Böck et al, 2006; Kwon et al, 2018)
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