Delimiting the Function of the C-Terminal Extension of the Escherichia coli [NiFe]-Hydrogenase 2 Large Subunit Precursor

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.

A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation

“…This conclusion is in contrast to in vitro experiments showing the interaction of the apo‐form of the large subunit of E. coli Hyd‐2 with the Hyp protein delivering the Fe(CN) 2 (CO) unit of the active site was abolished in the absence of the C‐terminal extension (Senger et al, 2017). However, there are strong indications that rather the N‐terminus of the large subunit is involved in the interaction with the Hyp machinery (Albareda, Buchanan, & Sargent, 2017; Kwon et al, 2018; Pinske, Thomas, Nutschan, & Sawers, 2019; Thomas et al, 2015). Just recently, the crystal structure of apo‐HyhL, the large subunit of the [NiFe]‐hydrogenase from T. kodakarensis , in complex with the nickel‐inserting maturase HypA has been resolved (Kwon et al, 2018).…”

“…Therefore, a major role of the C‐terminal extension might be providing indirectly the N‐terminus with sufficient flexibility to interact with the Hyp machinery. A role of the C‐terminal extension in enhancing structural flexibility to facilitate the interaction with Hyp proteins has also been proposed (Albareda, Pacios, & Palacios, 2019; Pinske et al, 2019). A function as a maturation facilitator would also explain why the removal of the C‐terminal extension does not necessarily lead to immature hydrogenase.…”

The large subunit of the regulatory [NiFe]-hydrogenase fromRalstonia eutropha– a minimal hydrogenase?

“…6 Recent experiments conducted to unravel the chaperone function of the C-terminal extension of the Escherichia coli [NiFe]hydrogenase 2 large subunit precursor support this assumption. 7 In the present study, we focused our attention on the large subunit of the regulatory hydrogenase (RH) from R. eutropha, which belongs to the small subgroup of [NiFe]-hydrogenases whose large subunits do not possess a C-terminal extension. 8 The RH contains an otherwise standard-like catalytic center (Fig.…”

Activation of a [NiFe]-hydrogenase-4 isoenzyme by maturation proteases

“…Early work showed that swapping of the E. coli HycE (Hyd-3) assembly peptide for that of HybC (Hyd-2) led to a ‘dead-end’ fusion protein that could not be processed by any maturation protease tested [ 43 ]. While more recently, swapping the HybC (Hyd-2) assembly peptide for that of HyaB (Hyd-1) did not lead to any changes in the requirement for the Hyd-2-specific protease (HybC) for maturation [ 42, 44 ].…”

“…It is becoming increasingly clear that the C-terminal assembly peptide may not be the key recognition motif for the protease [34,42]. Early work showed that swapping of the E. coli HycE (Hyd-3) assembly peptide for that of HybC (Hyd-2) led to a 'dead-end' fusion protein that could not be processed by any maturation protease tested [43].…”