Coordination of Synthesis and Assembly of a Modular Membrane-Associated [NiFe]-Hydrogenase Is Determined by Cleavage of the C-Terminal Peptide

Abstract

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...

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

“…Previous studies revealed that the genetic removal of the C‐terminal extension of the large subunit results in hydrogenase devoid of a functional NiFe active site (Massanz et al, 1997; Thomas et al, 2015). To test whether the absence of the C‐terminal extension of the membrane‐bound hydrogenase (MBH) of R. eutropha also leads to inactive protein, we deleted the sequence encoding the amino acid residues Val604‐Arg618 of the HoxG subunit.…”

“…They observed that the genetically processed large subunit of E. coli Hyd2 lacks the native NiFe cofactor but forms a complex with the small subunit. The resulting inactive Hyd2 was even accepted by the Tat translocation apparatus and appropriately inserted into the cytoplasmic membrane (Thomas et al, 2015). Therefore, we analyzed the catalytic activity and the cofactor content of the MBH proc purified from the membrane fraction of R. eutropha HP9 and compared the results with those of R. eutropha HP3, synthesizing native MBH.…”

“…The apo‐large subunit is usually synthesized with a C‐terminal extension comprising 3–68 amino acids (Greening et al, 2015), which is cleaved off by a specific endopeptidase once the complete NiFe site has been incorporated (Böck et al, 2006; Fritsch, Lenz, & Friedrich, 2013; Theodoratou, Huber, & Böck, 2005). Modifications of this C‐terminal extension, including amino acid exchanges, truncation (Theodoratou, Paschos, Mintz‐Weber, & Böck, 2000), or even complete removal (Massanz, Fernandez, & Friedrich, 1997; Senger, Stripp, & Soboh, 2017; Thomas, Muhr, & Sawers, 2015), by genetic engineering generally lead to the formation of inactive hydrogenase. Interestingly, while exchanges and truncations revealed a premature large subunit that was unable to form a complex with the small subunit, genetic removal of the entire extension allowed the formation of hydrogenase with canonical subunit composition.…”

The dual-function chaperone HycH improves assembly of the formate hydrogenlyase complex

“…In the processed HycE model structure, this Arg537 is buried within the protein, indicating a possible structural rearrangement after the proteolytic processing, as has been proposed for Hyd-2 [7,39]. The C-terminal extension of HycE itself has a total of 30% basic amino acids and HycH is predicted to be relatively acidic, however, with low conservation of these residues; thus, it is unlikely that the C-terminal extension is the interaction interface with HycH.…”

Biosynthesis of Salmonella enterica [NiFe]-hydrogenase-5: probing the roles of system-specific accessory proteins

“…The proteins we have chosen not to study, S. enterica HydD, HydE and HydI are respectively identified as: a maturation protease found in the operons of all [NiFe] MBH and comparable to the structurally characterized protein E. coli HybD [29,30]; a large subunit maturase similar to E. coli HypC, R. eutropha HoxL [31] and Rh. leguminosarum HupF [28], which have well characterised roles in enzyme assembly; and a HypA homolog [2], which is believed to be involved in the final step of incorporating nickel into the metallocentre [32].…”