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 (original) (raw)

1999, Journal of Biological Chemistry

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. 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 2؋[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 ʈ ʈ,Ќ Ќ ‫؍‬ 2.05, ϳ1.93, and g ʈ ʈ,Ќ Ќ ‫؍‬ 2.08, ϳ1.90, and respective slow (P1 ⁄2 ‫؍‬ 8 milliwatts) and fast (P1 ⁄2 ‫؍‬ 342 milliwatts) spin relaxation; (ii) two clusters exhibit similar intrinsic EPR spectra (g ʈ ʈ,Ќ Ќ ‫؍‬ 2.05, ϳ1.93) 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 ʈ ʈ,Ќ Ќ ‫؍‬ 2.08, ϳ1.90, with enhanced spin relaxation. In either case, our EPR data provide the first experimental evidence for the presence of two [4Fe-4S] ironsulfur clusters in the NQO9 subunit. The proton-translocating NADH-quinone (Q) 1 oxidoreductase is one of five enzyme complexes in the oxidative phosphorylation system that is located in the inner membrane of mitochondria and in the cytoplasmic membrane of bacteria. The mitochondrial enzyme is termed complex I, and the bacterial enzyme is called NDH-1. Both enzyme complexes catalyze the Reaction 1. NADH ϩ Q ϩ H ϩ ϩ nH ϩ ϩ NAD ϩ in 3 NAD ϩ ϩ QH 2 ϩ nH ϩ out (n ϭ 3-5)