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 activ ity under anaerobic growth conditions. The enzyme is a complex of a 93 -kDa polypeptide and a 16-kDa nitrate-oxidizable cytochrome c(552). Th e complex contains molybdenum; a fluorescent compound with spectral fe atures of a pterin derivative can be extracted. In contrast to the dis similatory membrane-bound nitrate reductases, the periplasmic nitrate reductase shows high specificity for nitrate as a substrate and is ins ensitive to inhibition by azide. The 93-kDa subunit exhibits immunolog ical cross-reactivity with the catalytic subunit of Rhodobacter capsul atus N22DNAR(+) periplasmic nitrate reductase. Mass spectrometric comp arisons of holo-cytochrome c(552) and apo-cytochrome c(552) demonstrat ed that the polypeptide bound two haem groups. Mediated redox potentio metry of the cytochrome indicated that the haem groups have reduction potentials (pH = 7.0) of approximately -15 mV and +80 mV. The function al significance of these potentials is discussed in relation to the pr oposed physiological role of the enzyme as a redox valve.