Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha.
The periplasmic nitrate reductase of Thiosphaera pantotropha has been purified from a mutant strain (M-6) that overproduces the enzyme activity under anaerobic growth conditions. The enzyme is a complex of a 93-kDa polypeptide and a 16-kDa nitrate-oxidizable cytochrome c552. The complex contains molybdenum; a fluorescent compound with spectral features of a pterin derivative can be extracted. In contrast to the dissimilatory membrane-bound nitrate reductases, the periplasmic nitrate reductase shows high specificity for nitrate as a substrate and is insensitive to inhibition by azide. The 93-kDa subunit exhibits immunological cross-reactivity with the catalytic subunit of Rhodobacter capsulatus N22DNAR+ periplasmic nitrate reductase. Mass spectrometric comparisons of holo-cytochrome c552 and apo-cytochrome c552 demonstrated that the polypeptide bound two haem groups. Mediated redox potentiometry of the cytochrome indicated that the haem groups have reduction potentials (pH = 7.0) of approximately -15 mV and + 80 mV. The functional significance of these potentials is discussed in relation to the proposed physiological role of the enzyme as a redox valve.
Berks
B C
BC
Department of Biochemistry, University of Oxford, England.
Richardson
D J
DJ
Robinson
C
C
Reilly
A
A
Aplin
R T
RT
Ferguson
S J
SJ
D002462:Cell Membrane / Q000201:enzymology; D006418:Heme / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008564:Membrane Potentials; D008970:Molecular Weight; D009154:Mutation; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D006418:Heme; D009565:Nitrate Reductases; D050901:Nitrate Reductase