Abstract
During anaerobic growth Escherichia coli synthesizes two membrane-associated hydrogen-oxidizing [NiFe]-hydrogenases, termed hydrogenase 1 and hydrogenase 2. Each enzyme comprises a catalytic subunit containing the [NiFe] cofactor, an electron-transferring small subunit with a particular complement of [Fe-S] (iron-sulfur) clusters and a membrane-anchor subunit. How the [Fe-S] clusters are delivered to the small subunit of these enzymes is unclear. A-type carrier (ATC) proteins of the Isc (iron-sulfur-cluster) and Suf (sulfur mobilization) [Fe-S] cluster biogenesis pathways are proposed to traffic pre-formed [Fe-S] clusters to apoprotein targets. Mutants that could not synthesize SufA had active hydrogenase 1 and hydrogenase 2 enzymes, thus demonstrating that the Suf machinery is not required for hydrogenase maturation. In contrast, mutants devoid of the IscA, ErpA or IscU proteins of the Isc machinery had no detectable hydrogenase 1 or 2 activities. Lack of activity of both enzymes correlated with the absence of the respective [Fe-S]-cluster-containing small subunit, which was apparently rapidly degraded. During biosynthesis the hydrogenase large subunits receive their [NiFe] cofactor from the Hyp maturation machinery. Subsequent to cofactor insertion a specific C-terminal processing step occurs before association of the large subunit with the small subunit. This processing step is independent of small subunit maturation. Using western blotting experiments it could be shown that although the amount of each hydrogenase large subunit was strongly reduced in the iscA and erpA mutants, some maturation of the large subunit still occurred. Moreover, in contrast to the situation in Isc-proficient strains, these processed large subunits were not membrane-associated. Taken together, our findings demonstrate that both IscA and ErpA are required for [Fe-S] cluster delivery to the small subunits of the hydrogen-oxidizing hydrogenases; however, delivery of the Fe atom to the active site might have different requirements.
Highlights
Iron-sulfur ([Fe-S]) clusters are ubiquitous prosthetic groups of many metalloenzymes in almost all life-forms and they have a variety of functions in diverse cellular processes
We have examined the biosynthesis of the hydrogen-oxidizing [NiFe]-hydrogenases of E. coli with respect to the potential involvement of the three A-type carrier (ATC) paralogues IscA, SufA and ErpA
Western blot analysis of extracts derived from the iscA mutant CP477 and the erpA mutant LL402 [8] lacked the small subunits of hydrogenase 2 (Fig. 4D) and hydrogenase 1 (Fig. 4B and data not shown)
Summary
Iron-sulfur ([Fe-S]) clusters are ubiquitous prosthetic groups of many metalloenzymes in almost all life-forms and they have a variety of functions in diverse cellular processes. IscA gene on plasmid piscA into CP477 restored the activities of both hydrogen-oxidizing enzymes, their association with the membrane fraction (Fig. 5A), as well as the appearance of the membrane-associated, processed HybC large subunit and the small subunit HybO (Fig. 5B) Similar results to those observed for CP477 (DiscA) were observed when the sub-cellular fractions of the erpA mutant LL401 were analyzed (data not shown). Extracts from the iscU mutant JW2513 showed no polypeptide, a result which would be consistent with a lack of the [Fe-S] cluster in HypD causing rapid turnover of the protein (see [13]) This result demonstrates that the Isc machinery is required for insertion of the [Fe-S] cluster into HypD, but that in contrast to the hydrogenase small subunits, IscA and ErpA show redundancy in cluster delivery to HypD
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