Abstract
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.
Highlights
Molecular properties of the NQO9 subunit of Paracoccus denitrificans NDH-1, which is predicted to contain 2؋[4Fe-4S] clusters, were investigated using recombinant expression techniques and EPR spectroscopy
These results suggest that the NQO9 subunit is more hydrophobic than other peripheral subunits (NQO1, NQO2, NQO3, NQO4, and NQO5), which have been expressed as soluble proteins in E. coli [22, 23, 26, 28]
The low quantity of the membrane-localized subunit did not allow us to characterize molecular properties of the subunit. These results led us to consider that the NQO9 subunit may contain particular sequences that are responsible for its hydrophobic properties
Summary
Q, quinone; NDH-1, bacterial protontranslocating NADH-quinone oxidoreductases; complex I, mitochondrial proton-translocating NADH-quinone oxidoreductases; PdNQO9, NQO9 subunit of P. denitrificans NDH-1; PCR, polymerase chain reaction; DTT, dithiothreitol; NTA, nitrilotriacetic acid; mW, milliwatt. The subunit contains 11 fully conserved cysteine residues among its homologues including one [4Fe-4S] cluster-binding motif. The NQO6 subunit and its mitochondrial counterpart, PSST subunit, have been identified as a specific target of structurally diverse inhibitors such as pyridaben, rotenone, and piericidin A [15] This finding suggests that the NQO6 subunit plays an important role in electron transfer step from cluster N2 to quinone. Chemical and physicochemical analyses of the reconstituted NQO9 subunit demonstrated that the subunit contains 2ϫ[4Fe-4S] clusters
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