We analyzed the involvement of
chaperonins GroES and GroEL in the biosynthesis of the three
hydrogenase isoenzymes, HYD1,
HYD2, and HYD3, of Escherichia coli. These hydrogenases are NiFe-containing, membrane-bound
enzymes composed of small and large subunits, each of which is proteolytically processed during biosynthesis. Total
hydrogenase activity was found to be reduced by up to 60% in groES and groEL thermosensitive mutant strains. This effect was specific because it was not seen for another oligomeric, membrane-bound metalloenzyme, i.e.,
nitrate reductase. Analyses of the single
hydrogenase isoenzymes revealed that a temperature shift during the growth of groE mutants led to an absence of HYD1 activity and to an accumulation of the precursor of the large subunit of HYD3, whereas only marginal effects on the processing of
HYD2 and its activity were observed under these conditions. A decrease in total
hydrogenase activity, together with accumulation of the precursors of the large subunits of
HYD2 and HYD3, was also found to occur in a
nickel uptake mutant (nik). The phenotype of this nik mutant was suppressed by supplementation of the growth medium with
nickel ions. On the contrary, Ni2+ no longer restored
hydrogenase activity and processing of the large subunit of HYD3 when the nik and groE mutations were combined in one strain. This finding suggests the involvement of these
chaperonins in the biosynthesis of a functional HYD3
isoenzyme via the incorporation of
nickel. In agreement with these in vivo results, we demonstrated a specific binding of GroEL to the precursor of the large subunit of HYD3 in vitro. Collectively, our results are consistent with
chaperonin-dependent incorporation of
nickel into the precursor of the large subunit of HYD3 as a prerequisite of its proteolytic processing and the acquisition of enzymatic activity.