Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha

Ben C. Berks, David J. Richardson, Carol Robinson, Ann Reilly, Robin T. Aplin, Stuart J. Ferguson

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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 languageEnglish
Pages (from-to)117-124
Number of pages8
JournalEuropean Journal of Biochemistry
Issue number1
Publication statusPublished - Feb 1994

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