INTERACTION OF THE HYDROGENASE ACCESSORY PROTEIN HYPC WITH HYCE, THE LARGE SUBUNIT OF ESCHERICHIA-COLI HYDROGENASE-3 DURING ENZYME MATURATION

Maturation of the large subunit of E. coli hydrogenase 3, HycE, requir es the action of seven accessory proteins. The HycI protease catalyses a C-terminal proteolytic cleavage of the large subunit, which was sho wn to result in a dramatic change in migration behavior of HycE in non denaturing PAGE. HypA, HypB, HypC, HypD, HypE, and HypF are required f or metallocenter assembly. A polyacrylamide gel system under nondenatu ring conditions was used for the investigation of any protein-protein interactions between HycE and the Hyp proteins. It revealed the existe nce of a complex between the precursor of HycE (pre-HycE) with one of the accessory proteins, namely HypC. HypC migrates in at least three d ifferent forms in nondenaturing PAGE, the appearance of one of which ( form 1) is strictly dependent on the presence of unprocessed HycE in t he extract. Overexpression of either hypC or hycE from a plasmid leads to an increased formation of this HypC-form 1. In two-dimensional pol yacrylamide gel electrophoresis with nondenaturing PAGE as the first a nd SDS-PAGE as the second dimension, this HypC form comigrates with pa rt of the pre-HycE protein. This comigration was also observed in anio n exchange chromatography. To analyze the pre-HycE-HypC complex in mor e detail, HypC was overproduced and purified. The purified protein was able to bind to pre-HycE in vitro. These results and also the finding that the processed form of HycE is not associated with HypC suggest t hat HypC binds to pre-HycE to keep it in a conformation accessible for metal incorporation.