Respiratory nitrate reductase from Paracoccus denitrificans

Abstract

1. The b-type haem centres of the three (a, fi and y) subunit nitrate reductase from Paracoccus denitrijkans have been analysed by redox potentiometry. Two components were identified with mid-point potentials + 95 mV and +210 mV.2. Washing, in the absence of Mg2+ ions, of cytoplasmic membrane vesicles from P. denitriJicans promoted selective release of nitrate reductase activity. The released enzyme was purified by chromatography and shown to contain a and fi, but not y polypeptides. A haem spectrum was absent, consistent with the lack of the y subunit. The CI and fi polypeptides of the water-soluble nitrate reductase had molecular masses that were identical to those of the detergent-purified enzyme and also of the nitrate reductase in cytoplasmic membranes. This observation, together with the failure of protease inhibitors to prevent release from the membrane, indicates that the release is not related to limited proteolysis of the a and/or polypeptides. The relative molecular mass of the watersoluble afi enzyme was estimated to be approximately 200 000.3. The water-soluble nitrate reductase was released from intact inverted cytoplasmic membrane vesicles as jugded by loss of NADH-NO; reductase activity and retention by the vesicles after washing of uncoupler-sensitive NADH-oxidase activity. These observations show that a and fi polypeptides, and therefore the active site for nitrate reduction, are located on the cytoplasmic side of the membrane.4. Attempts to reverse the nitrate reductase activity of the enzyme, using nitrite as reductant plus ferricyanide or chlorate as tested oxidants, were unsuccessful. The implications for the mechanism of the enzyme are discussed.The respiratory nitrate reductase catalyses the first step of bacterial denitrification. Despite this important role, relatively little is known about these enzymes from denitrifying organisms, although there has been fairly extensive study of the nitrate reductase from Escherichia coli, an organism which does not denitrify [l, 21. We recently showed that for the denitrifier Paracoccus denitrzjicans the nitrate reductase can be purified in the presence of detergent from cytoplasmic membranes in a form that has three polypeptides, designated a, p and y. These have similar molecular masses to the corresponding components of the E. coli enzyme [3]. A further similarity between the enzymes is that the y subunit has in each case been identified as the cytochrome subunit [l -31.Hackett and Bragg [4] have presented evidence that a partially purified nitrate reductase from E. coli contains two b-type haem centres that can be distinguished on the basis of redox potential. Such observations, together with the evidence that the b-type cytochrome of the bcl complex of mitochondria and photosynthetic bacteria contains two haem groups [51, prompted analysis, reported here, of this aspect of the purified nitrate reductase from P. denitrijkans.A pre-requisite for understanding the functioning of the nitrate reductase in P. denitrificans is the determination of the...

Heme Axial Ligation by the Highly Conserved His Residues in Helix II of Cytochrome b (NarI) of Escherichia coliNitrate Reductase A (NarGHI)

“…We have previously shown that the BV . ϩ :NO 3 Ϫ oxidoreductase activity appears to be independent of the presence of NarI (2,24). This prompted us to investigate whether the NarI-H56R mutation has any effect on the assembly or conformation of the NarH [Fe-S] clusters.…”

“…3 Comparison of the potentiometric titration data from the wild-type enzyme (Fig. 3) with the effect of the H56R mutation on the heme EPR line shape (Fig.…”

“…NarGHI is a complex of three nonidentical subunits, NarG (150 kDa), NarH (60 kDa), and NarI (25 kDa), in the ratio of 1:1:1 as predicted by the operon organization. This complex is arranged in two catalytic domains: the NarGH subunits constitute the cytoplasmic domain and NarI the membrane anchor domain (2,3). The NarGH domain contains four [Fe-S] centers (4,5) and a molybdenum cofactor which is the site of nitrate reduction by the enzyme (6).…”

Regulation of oxidative phosphorylation: The flexible respiratory network ofParacoccus denitrificans

“…The first reduction step in the process of denitrification is catalyzed by nitrate reductase (Nar), which converts nitrate to nitrite: NO~-+ 2H + + 2e----> NO~-+ HzO. This membrane-bound enzyme resembles the dissimilatory nitrate reductase of E. coil to a great extent (Ballard and Ferguson, 1988;Stewart, 1988). The enzyme consists of three different subunits (a, 13, and "y) and it is suggested that during nitrate reduction electrons from ubiquinol are passed to the b-type heroes in the -,/-subunit and subsequently via iron-sulfur centers in the 13-subunit to the molybdenum cofactor, which is located in the a-subunit (Fig.…”

Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617

“…With the exception of the eukaryotic nitrate reductases, which are part of the sulfite oxidase family, all the prokaryotic nitrate reductases belong to the dimethyl sulfoxide (DMSO) reductase family of Mo-containing enzymes [7]. The respiratory nitrate reduction pathway involves the membrane-bound respiratory nitrate reductase (Nar), which has been isolated from a variety of organisms, including a few well-studied bacteria [8][9][10][11][12][13][14]. Nars are involved in the redox loop generating the proton motive force across biomembranes [15].…”