Abstract
Hydrogenases catalyze the reversible activation of dihydrogen. We have previously demonstrated that the purified hydrogenase from the nitrogen-fixing microorganism Azotobacter vinelandii is an alpha beta dimer (98,000 Da) with subunits of 67,000 (alpha) and 31,000 (beta) daltons and that this enzyme contains iron and nickel. The enzyme can be purified anaerobically in the presence of dithionite in a fully active state that is irreversibly inactivated by exposure to O2. Analysis of this hydrogenase by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) following boiling in SDS yields two protein staining bands corresponding to the alpha and beta subunits. However, when this enzyme was treated with SDS (25-65 degrees C) for up to 30 min under anaerobic/reductive conditions and then analyzed by anaerobic SDS-PAGE, a protein staining band corresponding to an apparent molecular mass of 58,000 Da was observed that stained for hydrogenase activity. Analysis of the 58,000-Da activity staining band by a Western immunoblot or a second aerobic SDS-polyacrylamide gel revealed that this protein actually consisted of both the alpha and beta subunits. Thus, the activity staining band (apparent 58,000 Da) represents the 98,000-Da dimer migrating abnormally on SDS-PAGE. Treatment of the anaerobically purified hydrogenase with SDS under aerobic conditions or under anaerobic conditions with electron acceptors prior to electrophoresis resulted in no activity staining band and the separated alpha and beta subunits. A. vinelandii hydrogenase was also purified under aerobic conditions in an inactive O2 stable form that can be activated by removal of oxygen followed by addition of reductant. This enzyme (as isolated), the activated form, and the reoxidized form were analyzed for their stability toward denaturation by SDS. We conclude that the dissociation of the A. vinelandii hydrogenase subunits in SDS is controlled by the redox state of the enzyme suggesting an important role of one or more redox sites in controlling the structure of this enzyme.
Highlights
Hydrogenases catalyze the reversible activation of iron as partof iron-sulfur clusters, the numbers and dihydrogen
In an attempt to characterize the physical and catalytic properties of the large and small subunits of dimeric hydrogenases,the original observation of Kowand Burris[10] was re-examined in light of the dimeric structure of the A. vinelandii hydrogenase.In thiswork, we present evidence that anaerobically purified A. vinelandii hydrogenase can migrate as the a@ dimer (98,000 Da) at an apparent molecular mass of 58,000 Daon sodium dodecylsulfate (SDS) gel electrophoresis under anaerobic/ reductive conditions and that thibsand retainsactivity
If hydrogenase was treated with 3.5% (w/v) SDS under anaerobic conditions a t various temperatures and analyzed on an anaerobic SDS-polyacrylamide gel, a single hydrogenase activity staining band was observed at an apparent M, of 58,000 (Fig. l B, lanes 1-3)
Summary
We have shown in a previous work that the membranebound hydrogenase purified from A. vinelandii is an a@ dimer with a native molecular weight of 98,000 [9] Analysis of this protein by SDS-PAGE following boiling in 3.5% (w/v) SDS yields noactivity staining bands and two protein staining bands, one corresponding to the a subunit (67,000 Da) and one to theB subunit (31,000Da) (Fig. LA, lane 7). If hydrogenase was treated with 3.5% (w/v) SDS under anaerobic conditions (argon, 2 mM dithionite) a t various temperatures and analyzed on an anaerobic SDS-polyacrylamide gel, a single hydrogenase activity staining band was observed at an apparent M , of 58,000 (Fig. l B , lanes 1-3) This bandwas the major protein-staining band (Fig. LA, lanes 1-3). This suggests that the active protein with an apparentM , of 58,000 on SDS gels consists of both a and @ subunits
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