Expression of the 25-kilodalton iron-sulfur subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans
- 18 January 1994
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 33 (2) , 494-499
- https://doi.org/10.1021/bi00168a014
Abstract
The energy-transducing NADH-ubiquinone (Q) oxidoreductase of Paracoccus denitrificans is composed of 14 dissimilar subunits and contains at least four iron-sulfur clusters [Yagi, T. (1993) Biochim. Biophys. Acta 1141, 1-17]. The complete DNA sequence of the gene cluster encoding the energy-transducing NADH-Q oxidoreductase of P. denitrificans has been determined. This paper reports the expression of the 25-kilodalton (kDa) (NQO2) subunit of the P. denitrificans enzyme complex in Escherichia coli and the characterization of the iron-sulfur cluster bound to the expressed subunit. The 25-kDa subunit was expressed in the cytoplasmic phase but not in the membrane fraction of E. coli cells and then purified using an affinity nickel chelation column. The purified subunit contains 1.44 mol of non-heme iron and 1.33 mol of acid-labile sulfide/mol of subunit. EPR analysis of the reduced form of this subunit indicates that the expressed subunit contains a single binuclear [2Fe-2S] cluster. This cluster exhibits a spectrum of rhombic symmetry with g values of gx,y,z = 1.913, 1.942, and 1.996, which is very similar to the spectrum of the [2Fe-2S] cluster in the resolved flavoprotein II subfraction (subunit 24 + 9 kDa) of bovine heart complex I [Ragan, C. I., Galante, Y. M., Hatefi, Y., & Ohnishi, T. (1982) Biochemistry 21, 590-594; Ohnishi, T., Ragan, C. I., & Hatefi, Y. (1985) J. Biol. Chem. 260, 2782-2788]. The assignment of the binuclear iron-sulfur cluster of the 25-kDa subunit to an EPR-visible iron-sulfur cluster in the Paracoccus NADH-Q oxidoreductase in situ is discussed.Keywords
This publication has 32 references indexed in Scilit:
- Assembly of the Rieske iron-sulfur subunit of the cytochrome bc1 complex in the Escherichia coli and Rhodobacter sphaeroides membranes independent of the cytochrome b and c1 subunitsBiochemistry, 1993
- Genetics of Paracoccus denitrificansFEMS Microbiology Letters, 1993
- Characterization of the 25-kilodalton subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans: sequence similarity to the 24-kilodalton subunit of the flavoprotein fraction of mammalian complex IBiochemistry, 1991
- The NADH-binding subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans: gene cloning and deduced primary structureBiochemistry, 1991
- Identification of the NADH-binding subunit of energy-transducing NADH-quinone oxidoreductase (NDH-1) of thermus thermophilus HB-8Biochemical and Biophysical Research Communications, 1991
- Identification of the NADH-binding subunit of NADH-ubiquinone oxidoreductase of Paracoccus denitrificansBiochemistry, 1990
- Cytochromecoxidase in prokaryotesFEMS Microbiology Letters, 1987
- A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopyBiochimica et Biophysica Acta (BBA) - Bioenergetics, 1980
- Bioenergetic studies on Paracoccus denitrificansTrends in Biochemical Sciences, 1980
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970