Open AccessPurification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha
Author(s) -
BERKS Ben C.,
RICHARDSON David J.,
ROBINSON Carol,
REILLY Ann,
APLIN Robin T.,
FERGUSON Stuart J.
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.tb18605.x
Subject(s) - periplasmic space , nitrate reductase , chemistry , cytochrome , biochemistry , heme , rhodobacter , protein subunit , enzyme , mutant , escherichia coli , gene
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.