MATURATION OF THE LARGE SUBUNIT (HYCE) OF ESCHERICHIA-COLI HYDROGENASE 3 REQUIRES NICKEL INCORPORATION FOLLOWED BY C-TERMINAL PROCESSING ATARG537

Purification of the large subunit, HYCE, of Escherichia coli hydrogena se 3 revealed that it is a nickel-containing polypeptide, which is sub ject to C-terminal proteolytic processing. This processing reaction co uld be performed in vitro with partially purified components, yielding a low-molecular mass C-terminal peptide which was resolved in a Trici ne/SDS/polyacrylamide gel. N-terminal sequencing of this peptide revea led that proteolytic cleavage occurred at the C-terminal side of the a rginine residue at position 537, which corresponds to the histidine re sidue in the highly conserved motif, DPCXXCXXH, of other (NiFe) hydrog enases thought to be involved in active site nickel coordination. Nick el-containing HYCE precursor for in vitro processing, was partially pu rified from strain HD708 (Delta hycN) in the presence of the reducing agent dithiothreitol. Using 2-mercaptoethanol instead of dithiothreito l provided pure precursor, which was, however, no longer susceptible t o in vitro processing; it proved to be devoid of nickel indicating tha t nickel incorporation into the HYCE precursor is a prerequisite for p rocessing. This conclusion was supported by the finding that HYCE prec ursor from strain HD708 (Delta hycH) chromatographed with radioactivit y from Ni-63 incorporated in vivo and could be processed in vitro, whe reas HYCE precursor from strain BEF314 (Delta hypB-E) lacking the nick el insertion system appeared to be devoid of nickel and was not sensit ive to in vitro processing.