Evidence for an oxygen-sensitive iron-sulfur cluster in an immature large subunit species of Escherichia coli [NiFe]-hydrogenase 2.
Endoprotease-specific C-terminal processing is required to complete the maturation of the large subunit of [NiFe]-hydrogenases. This happens only after synthesis and insertion of the NiFe(CN)(2)CO cofactor by the Hyp maturases has occurred. It is assumed that in the absence of maturation the unprocessed species of the large subunit lacks cofactors. In this study we isolated a variant of the hydrogenase 2 large subunit, HybC, containing a fused C-terminal pentapeptide. The polypeptide could not be processed and was unable to associate with the small subunit to deliver an active enzyme. The His(6)-HybC variant protein isolated was brown and had sub-stoichiometric amounts of an oxygen-sensitive Iron-sulfur cluster, which could be chemically reconstituted to a [4Fe-4S] cluster. This cluster was coordinated by the conserved cysteinyl residues that normally ligate the NiFe(CN)(2)CO cofactor. Our findings provide evidence for temporary promiscuity of cofactor-binding sites.
Institute for Biology/Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany.
Basem
B
Soboh
Martin
M
Kuhns
Mario
M
Braussemann
Mandy
M
Waclawek
Enrico
E
Muhr
Antonio J
AJ
Pierik
R Gary
RG
Sawers
D019878:Carboxyl and Carbamoyl Transferases / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D010100:Oxygen / Q000737:chemistry*
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; C487901:IscA protein, E coli; D009842:Oligopeptides; D010088:Oxidoreductases; C043296:nickel-iron hydrogenase; C029746:uptake hydrogenase; D006864:Hydrogenase; D019878:Carboxyl and Carbamoyl Transferases; C504838:hypF protein, E coli; D010100:Oxygen