title: Hydrodynamic, structural and magnetic properties of Megasphaera elsdenii Fe hydrogenase reinvestigated.
abstract: Megasphaera elsdenii hydrogenase has been purified to homogeneity using an FPLC procedure as the final step. The protein gives a single band in SDS/PAGE with an apparent molecular mass of 57-59 kDa. There is no second hydrogenase activity in the soluble fraction of M. elsdenii. The hydrodynamics of the enzyme have been compared to those of the two-subunit Fe hydrogenase from Desulfovibrio vulgaris (Hildenborough) in the analytical ultracentrifuge using the absorption of the intrinsic iron-sulfur clusters as the monitor. Sedimentation-velocity experiments indicate the M. elsdenii enzyme (s20,w = 4.95 S) to be essentially globular, while the D. vulgaris enzyme (s20,w = 4.1 S) has a less symmetric shape. From the sedimentation equilibrium measurements under a variety of conditions an average molecular mass is calculated of 58 kDa (M. elsdenii) and 54 kDa (D. vulgaris), respectively. Pure, maximally active M. elsdenii hydrogenase has A405/A280 = 0.36 and has a specific H2-production activity of 400 mumol H2.min-1.(mg protein)-1 at 30 degrees C and pH 8.0. The enzyme contains some 13-18 iron and acid-labile sulfur ions/58-kDa monomer. Eight of these Fe-S are present as two electron-transferring ferredoxin-like cubanes with Em approximately greater than -0.3 V, as indicated by pH-dependent EPR spectroscopy on the H2-reduced enzyme. In the (re)oxidized state the remainder iron gives rise to a single S = 1/2 rhombic EPR signal. Hydrogen-production activity, content of remainder iron and rhombic EPR signal intensity are mutually correlated. Purified hydrogenase appears to exist as a mixture of fully active holoenzyme and inactive protein still carrying the two cubanes but deficient in active-site iron.
authors.affiliation: Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Filipiak
authors.forename: W R
authors.initials: WR
authors.lastname: Hagen
authors.forename: C
authors.initials: C
authors.lastname: Veeger
mesh_terms: D001421:Bacteria, Anaerobic / Q000201:enzymology*; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase / Q000302:isolation & purification*; D007501:Iron / Q000032:analysis; D012995:Solubility; D014461:Ultracentrifugation
chemical_list: D007501:Iron; D006864:Hydrogenase