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 c(552). 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 c(552) and apo-cytochrome c(552) 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.