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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.

Original publication

DOI

10.1111/j.1432-1033.1994.tb18605.x

Type

Journal article

Journal

Eur J Biochem

Publication Date

15/02/1994

Volume

220

Pages

117 - 124

Keywords

Cell Membrane, Heme, Hydrogen-Ion Concentration, Mass Spectrometry, Membrane Potentials, Molecular Weight, Mutation, Nitrate Reductase, Nitrate Reductases, Oxidation-Reduction, Paracoccus denitrificans, Substrate Specificity