Characterization of the putative 2x[4Fe-4S]-binding NQO9 subunit of the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans. Expression, reconstitution, and EPR characterization.
Molecular properties of the NQO9 subunit of Paracoccus denitrificans NDH-1, which is predicted to contain 2x[4Fe-4S] clusters, were investigated using recombinant expression techniques and EPR spectroscopy. The full-length form of NQO9 subunit co-expressed with thioredoxin in Escherichia coli at ambient temperature was found dominantly in the cytoplasmic membrane with low amplification. Genetic deletion of relatively hydrophobic and less conserved N-terminal stretches (30 or 40 amino acid residues long) of the NQO9 subunit resulted in the overexpression of the truncated soluble form of the subunit in a high yield in the cytoplasm. The purified soluble form of the NQO9 subunit contained only a small quantity of Fe and S(2-) (2.0-2.2 mol each per mol of subunit). However, the iron-sulfur content was considerably increased by in vitro reconstitution. The reconstituted NQO9 subunit contained 7.6-7.7 mol each of Fe and S(2-) per molecule and exhibited optical absorption spectra similar to those of 2x[4Fe-4S] ferredoxins. Two sets of relatively broad axial-type EPR signals with different temperature dependence and power saturation profile were detected in the dithionite-reduced preparations at a low temperature range (8-18 K). Due to a negative shift (<600 mV) of the apparent redox midpoint potential of the iron-sulfur clusters in the soluble form of the truncated NQO9 subunit, the following two possible cases could not be discriminated: (i) two sets of EPR signals arise from two distinct species of tetranuclear iron-sulfur clusters with two intrinsically different spectral parameters g(, perpendicular) = 2.05, approximately 1.93, and g(parallel, perpendicular) = 2.08, approximately 1.90, and respective slow (P((1)/(2)) = 8 milliwatts) and fast (P((1)/(2)) = 342 milliwatts) spin relaxation; (ii) two clusters exhibit similar intrinsic EPR spectra (g(parallel, perpendicular) = 2.05, approximately 1.93) with slow spin relaxation. When both clusters in the same subunit are concomitantly paramagnetic, their spin-spin interactions cause a shift of spectra to g(parallel, perpendicular) = 2.08, approximately 1.90, with enhanced spin relaxation. In either case, our EPR data provide the first experimental evidence for the presence of two [4Fe-4S] iron-sulfur clusters in the NQO9 subunit.
Yano
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Division of Biochemistry, Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Magnitsky
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Sled'
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Ohnishi
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Yagi
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D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D017136:Ion Transport; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011522:Protons; D011808:Quinone Reductases / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D007506:Iron-Sulfur Proteins; D011522:Protons; D011994:Recombinant Proteins; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases