Nickel serves as a substrate recognition motif for the endopeptidase involved in hydrogenase maturation

Abstract

The interaction of the hydrogenase maturation endopeptidase HycI with its substrate, the precursor of the large subunit, was studied. Replacement of conserved amino-acid residues in HycI, which have been shown to bind a cadmium ion from the crystallization buffer in crystals of HybD (endopeptidase for hydrogenase 2), abolished or strongly reduced processing activity. Atomic absorption spectroscopy of purified HycI and HybD proteins showed the absence of nickel. In vitro processing assays showed that the reaction requires nickel to be bound to the precursor and the protease does not have a function in nickel delivery to the substrate. Radioactive labelling of cells with 63 Ni, devoid of endopeptidase, resolved several forms of the precursor which are possibly intermediates in the maturation pathway. It is concluded that the endopeptidase uses the metal in the large subunit of [NiFe]-hydrogenases as a recognition motif.Keywords: endopeptidase; hydrogenase maturation; nickel; processing.Genetic analysis of the maturation of active [NiFe]-hydrogenases revealed that the activities of at least seven gene products are involved in the process. These are the products of the so-called hyp genes [1,2], which include a specific chaperone (HypC) [3], a proposed nickel-donating GTPase (HypB) [4,5] and an endopeptidase that proteolytically removes a C-terminal extension from the precursor form of the large hydrogenase subunit (reviewed in [6]). The endopeptidases specific for hydrogenases 3 (HycI) and 2 (HybD) have been purified and an in vitro processing system has been established for the maturation of hydrogenase 3 [7,8].Major progress in the understanding how the endopeptidase recognizes its substrate came from the determination of the crystal structure of HybD [9]. It crystallizes as a complex with a cadmium ion from the crystallization buffer; the metal is pentacoordinated by three amino-acid side chains (Glu16, Asp62 and His93) and a water molecule in a pseudo-tetragonal arrangement. We proposed that the cadmium site marks the nickel binding site by which the protease interacts with the nickel-containing precursor of the large subunit. A model has been postulated which implies that iron and nickel are ligated separately to the apoprotein and are assembled into the complete heterobinuclear metal center after cleavage of the C-terminus [6]. Alternatively, the cadmium (i.e. nickel)-binding site may indicate that the endopeptidase is a nickel-dependent metalloprotease or that it is involved in metal transfer and incorporation into the hydrogenase large subunit. The observations that the precursor of the large subunit which accumulates in a protease HycI-deficient strain contains nickel and that cleavage appears to require prior nickel incorporation [8] argue against those functions but do not rule them out completely. The reason is that both in vivo and in vitro the precursor of the large subunit occurs in multiple folding states [3] and cannot be converted stoichiometrically into the mature form. In this communication, ...

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

“…However, we cannot currently explain the apparent double bands of the HycE protein under conditions where cofactor insertion is incomplete, e.g. in the ΔnikC and ΔhypC strains, and which, thus, should not result in HycI-dependent processing of HycE [36]. Both forms of HycE, however, also elute after the HycH Strep purification and only the lower band is visible when HycI is co-expressed.…”

Proteolytic inventory of Thermococcus kodakaraensis

“…Larger subunit contains a metal binding site and once metal binds, the subunit is cleaved near the C-terminal by hydrogenase maturation peptidases. Hydrogenase maturation protease Hyc1 is a member of this family which cleaves an Arg-Met bond immediately following the metal binding site of the hydrogenase (Theodoratou et al, 2000). Hyc1 from E. coli have been characterized and found to be an aspartate peptidase (Rossmann et al, 1995).…”

Solution Structure and Backbone Dynamics of an Endopeptidase HycI from scherichia coli

“…Metal Ion Binding-Extensive investigations into the mechanisms of maturation of HycE have been carried out, and it was shown that nickel ion plays a key role in the substrate recognition of endopeptidases (10,12). We performed the titration experiments using Ni 2ϩ and Cd 2ϩ monitored by two-dimensional 15 N-edited HSQC spectra.…”

“…Previous in vivo and in vitro experiments demonstrated that nickel ion (Ni 2ϩ ) is essential for the recognition and cleavage of the large subunit by the endopeptidase (11,12). Furthermore, the C terminus of the pre-large subunit may have interaction with other parts of the protein to form an open conformation amenable to nickel and endopeptidase recognition before it is cleaved (13)(14)(15).…”