The iron‐sulfur clusters in the two related forms of mitochondrial NADH: ubiquinone oxidoreductase made by Neurospora crassa

Abstract

Two related forms of the respiratory-chain complex, NADH : ubiquinone oxidoreductase (Complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large, piericidin-A-sensitive form, which consists of some 23 different nuclear-and 6 -7 mitochondrially encoded subunits. Cells grown in the presence of chloramphenicol make a small, piericidin-A-insensitive form which consists of only = 13 nuclearencoded subunits. The subunits of the small form are either identical or similar to nuclear-encoded subunits of the large form. The iron-sulfur clusters in these two forms of Complex I are characterized by redox potentiometry and EPR spectroscopy. The large form of Complex I contains four EPR-detectable iron-sulfur clusters, N1, N2, N3 and N4, with the spin concentration of the individual clusters equivalent to the flavin concentration, similar to the mammalian counterparts. The small Complex I contains clusters N1, N3 and N4, but it is devoid of cluster N2. A model of the electron-transfer route through the large form of Complex I has been derived from these findings and an evolutionary pathway which leads to the emergence of large Complex I is discussed.The mitochondrial respiratory-chain Complex I, NADH : ubiquinone oxidoreductase, links the transfer of two electrons from NADH to ubiquinone with the translocation of four or five protons across the mitochondrial inner membrane (for reviews see [I, 21). The enzyme is most complicated and consists of some 30 different subunits of which seven in human [3, 41 and at least six in the fungus N. crassa [5,61 are encoded by mitochondrial NADH dehydrogenase genes (ndh genes) and translated on mitochondrial ribosomes. The other subunits are nuclear encoded and imported from the cytoplasm. One FMN, four or five EPR-detectable iron-sulfur clusters [7, 81 and at least one bound form of ubiquinone participate in the electron transfer through the enzyme [9-111. Because of this enormous complexity, the NADH : ubiquinone oxidoreductase has remained the least understood among the proton-translocating respiratory-chain complexes of mitochondria. Therefore, attempts have been made to characterize related, putatively less complicated NADH : ubiquinone oxidoreductases in bacteria [I2 -191.Recently Friedrich et al. [20] have found that in chloramphenicol-poisoned N . crassa a smaller form of the NADH:ubiquinone oxidoreductase is made in place of the large Complex I normally present. This small Complex I consists of only = 13 subunits, all of which are nuclear encoded and synthesized in the cytoplasm. Immunoblotting and peptide mapping of the subunits found in the two forms of Complex I suggest that they are either identical or similar toCorrespondence to

The respiratory‐chain NADH dehydrogenase (complex I) of mitochondria

“…The redox potential values for the clusters N-1, N-2, N-3, N-4 in the N . crassa enzyme are -330, -150, -230, and -300 mV, respectively [18]. Most authors believe that the clusters are present at concentrations comparable to FMN, but Albracht and his group [21, 221 consistently report cluster N-1 to be present at half the concentration of the other clusters.…”

“…In the N . crassa enzyme no experimental evidence for two species of cluster N-1 were found [18]. There is EPR spectroscopic evidence for a protein-bound ubisemiquinone in NADH dehydrogenase, the signal of which is partially sensitive to rotenone [17, 231. Substrate channeling of NADH from mitochondrial matrix dehydrogenases to NADH dehydrogenase has been reported to occur by transient enzyme -enzyme binding [24].…”

“…The EPR spectra examined by the authors can, however, be interpreted as indicating a loss of only cluster N-2 under conditions of rotenone insensitivity 1181. N-2 [18]. Table 1).…”

“…For extensive reviews the readers are referred to [I 3 -151. Clearly established in the mammalian [lo, [13][14][15][16][17] and fungal enzyme [18] are the [2Fe-2S] cluster N-1 and the [4Fe-4S] clusters N-2, N-3 and N-4. In the mammalian NADH dehydrogenase, clusters N-1, N-3 and N-4 form an isopotential group with midpoint redox potentials (Em,,) around -250 mV.…”

The Iron−Sulfur Clusters 2 and Ubisemiquinone Radicals of NADH:Ubiquinone Oxidoreductase Are Involved in Energy Coupling in Submitochondrial Particles

“…The peripheral domain contains most of the redox groups, namely, FMN and the Fe-S clusters 1, 3, and 4 (Wang et al, 1991). There are two pathways of protein assembly, independently leading to the two major parts of the complex, the hydrophilic peripheral domain and the hydrophobic membrane domain, which then combine to form Complex I (Friedrich et al, 1989;Wang et al, 1991;Weiss et al, 1991).…”