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

Sven Hartmann,Oliver Lenz,Giorgio Caserta,Stefan Frielingsdorf

doi:10.1002/mbo3.1029

Abstract

[NiFe]‐hydrogenases catalyze the reversible conversion of molecular hydrogen into protons end electrons. This reaction takes place at a NiFe(CN)2(CO) cofactor located in the large subunit of the bipartite hydrogenase module. The corresponding apo‐protein carries usually a C‐terminal extension that is cleaved off by a specific endopeptidase as soon as the cofactor insertion has been accomplished by the maturation machinery. This process triggers complex formation with the small, electron‐transferring subunit of the hydrogenase module, revealing catalytically active enzyme. The role of the C‐terminal extension in cofactor insertion, however, remains elusive. We have addressed this problem by using genetic engineering to remove the entire C‐terminal extension from the apo‐form of the large subunit of the membrane‐bound [NiFe]‐hydrogenase (MBH) from Ralstonia eutropha. Unexpectedly, the MBH holoenzyme derived from this precleaved large subunit was targeted to the cytoplasmic membrane, conferred H2‐dependent growth of the host strain, and the purified protein showed exactly the same catalytic activity as native MBH. The only difference was a reduced hydrogenase content in the cytoplasmic membrane. These results suggest that in the case of the R. eutropha MBH, the C‐terminal extension is dispensable for cofactor insertion and seems to function only as a maturation facilitator.

Highlights

The reversible conversion of molecular hydrogen (H2) into protons and electrons is catalyzed by the enzyme hydrogenase (Lubitz, Ogata, Rüdiger, & Reijerse, 2014)
The role of the C-terminal extension in cofactor insertion, remains elusive. We have addressed this problem by using genetic engineering to remove the entire C-terminal extension from the apo-form of the large subunit of the membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha
The MBHproc of R. eutropha is the first example of a [NiFe]hydrogenase that is equipped with a NiFe catalytic center, the C-terminal extension of the large subunit was genetically removed

Summary

| INTRODUCTION

The reversible conversion of molecular hydrogen (H2) into protons and electrons is catalyzed by the enzyme hydrogenase (Lubitz, Ogata, Rüdiger, & Reijerse, 2014). 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 This phenomenon has been observed for the soluble, NAD+-reducing [NiFe]hydrogenase of R. eutropha (Massanz et al, 1997) and, more recently, for membrane-bound [NiFe]-hydrogenase 2 (Hyd-2) from Escherichia coli (Thomas et al, 2015). Nickel-free Hyd-2 was shown to lack the CN and CO ligands of the Fe(CN)2(CO) moiety, which are detectable by infrared spectroscopy (Senger et al, 2017) These observations suggest an essential role of the C-terminus in active site maturation. We deleted the C-terminal extension of HoxG by genetic engineering and show that the resulting truncated version of HoxG still receives a NiFe(CN)2(CO) cofactor and forms, together with the corresponding HoxK subunit, catalytically active MBH

| MATERIALS AND METHODS

Findings

| DISCUSSION