Isolation and characterization of the pyruvate-ferredoxin oxidoreductase from the sulfate-reducing bacterium Desulfovibrio africanus.
We report the first purification and characterization of a pyruvate-ferredoxin oxidoreductase (POR) from a sulfate-reducing bacterium, Desulfovibrio africanus. The enzyme as isolated is highly stable in the presence of oxygen and exhibits a specific activity of 14 U/mg. D. africanus POR is a 256 kDa homodimer which contains thiamine pyrophosphate (TPP) and iron-sulfur clusters. EPR spectroscopic study of the enzyme indicates the presence of three [4Fe-4S]2+/1- centers/subunits. The midpoint potentials of the three centers are -390 mV, -515 mV and -540 mV. The catalytic mechanism of POR involves a free radical intermediate which disappears when coenzyme A is added. This behaviour is discussed in terms of an electron-transport chain from TPP to the acceptor. The enzyme activated by dithioerythritol shows an exceptionally high activity compared with other mesophile PORs and becomes very sensitive to oxygen in contrast to the enzyme before activation. The comparison of EPR spectra given by the as isolated and activated enzymes shows that neither the nature, nor the arrangement of FeS centers are affected by the activation process. D. africanus ferredoxins I and II are involved as the physiological electron carriers of the enzyme. POR was shown to be located in the cytoplasm by immunogold labelling.
Pieulle
L
L
Unité de Bioénergétique et Ingénierie des Protéines, CNRS, Marseille, France.
Guigliarelli
B
B
Asso
M
M
Dole
F
F
Bernadac
A
A
Hatchikian
E C
EC
D000596:Amino Acids / Q000032:analysis; D003901:Desulfovibrio / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007658:Ketone Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D050743:Pyruvate Synthase; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000032:analysis
D000596:Amino Acids; D005288:Ferredoxins; D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase; D013835:Thiamine Pyrophosphate