Structural rearrangements in active and inactive forms of hydrogenase from Thiocapsa roseopersicina.
The electrophoretic behavior of Thiocapsa roseopersicina hydrogenase on sodium dodecyl sulfate gels demonstrates that the protein exists in two active forms, A1 and A2, which may be interconverted. Each of these forms has a characteristic electrophoretic mobility and differs in its sensitivity to O2. Form A1 is O2-labile and converts to A2 under O2. Form A2 is less sensitive to O2 and may be converted into A1 under H2 atmosphere. Both active forms are present in aerobically isolated samples. Because the proteins are still active on 15% sodium dodecyl sulfate gels, they are not completely denatured, and the apparent molecular masses do not necessarily represent the true molecular masses of the enzymes. A1 has an Rf = 0.19, corresponding to an apparent molecular mass of 90 kDa, and A2 has an Rf = 0.35, corresponding to an apparent molecular mass of 49 kDa. A sedimentation equilibrium centrifugation study of the active enzyme shows that the holoenzyme has a molecular mass of 98 kDa. Form A2 may be separated into two subunits of molecular mass of 64 kDa and 34 kDa, respectively. Thus, form A2 represents the holoenzyme with a true molecular mass of 98 kDa. Amino acid compositions and N-terminal amino acid sequences of the A2 protein and these subunits are consistent with a heterodimeric holoenzyme. The relationship between the conformational changes detected in this study and a three-state scheme proposed on the basis of EPR spectroscopic studies of the metal-containing cofactors present in the enzyme is also discussed.
Kovács
K L
KL
Institute of Biophysics, Biological Research Center, Szeged, Hungary.
Tigyi
G
G
Thanh
L T
LT
Lakatos
S
S
Kiss
Z
Z
Bagyinka
C
C
D000596:Amino Acids / Q000737:chemistry; D002841:Chromatiaceae / Q000201:enzymology* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D006864:Hydrogenase / Q000235:genetics*; D010449:Peptide Mapping
D000596:Amino Acids; D006864:Hydrogenase