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.