At this temperature cluster N1 can be measured selectively avoiding background effects from other overlapping iron-sulfur clusters, like iron-sulfur cluster S3 of complex II. To compensate for different complex I concentrations all spectra were normalized to the N1 g z signal intensity of the parental enzyme at 40 K (not shown).
In the second batch of complex I from mutant D99N, the presence of iron-sulfur cluster N2 was clearly evident from EPR spectra recorded at 12 K and the g z value for iron-sulfur cluster N2 was not shifted, indicating that the mutation had not influenced ligand geometry ( Fig. This structural instability was also evident from the fact that in the first complex I preparation from this mutant, cluster N2 was not detectable by EPR spectroscopy whereas the spectra of all other visible clusters were unaffected. The observation of decreased temperature stability of NADH:HAR oxidoreductase activity in D99N membranes was consistent with this interpretation (data not shown). As the content of complex I in membranes was only slightly lower that in the parental strain, the low purification yield suggested a somewhat lower stability of complex I in mutant D99N. Affinity purification of complex I from mutant D99N was successful, but the yield in two independent purification experiments was only 25% as compared with the parental strain. Due to this very low activity, determination of K m, V max, and I 50 values for complex I inhibitors was not possible. Changing this carboxylate into its corresponding amide asparagine resulted in almost complete loss (<5% residual activity) of dNADH:DBQ oxidoreductase activity in the presence of saturating amounts of both substrates. The results are discussed in the light of the structural similarities to the homologous small subunit of water-soluble hydrogenases.Ĭodon 99 in the NUKM open reading frame translates into a fully conserved aspartic acid ( Fig. This excludes all conserved acidic residues in the PSST subunit as fourth ligands of this redox center. Mutations of other conserved acidic residues had less dramatic effects on catalytic activity and did not prevent assembly of iron-sulfur cluster N2. In terms of complex I catalytic activity, almost identical results were obtained when the aspartates were individually changed to glutamates or to glycines.
In both cases complex I was stably assembled but electron paramagnetic resonance spectra of the purified enzyme showed a reduced N2 signal (about 50%). Mutants D99N and D115N had only 5 and 8% of the wild type catalytic activity, respectively. To explore the function of this subunit we have generated site-directed mutants of all eight highly conserved acidic residues in the Yarrowia lipolytica homologue, the NUKM protein. Subunit PSST is the most likely carrier of iron-sulfur cluster N2, which has been proposed to play a crucial role in ubiquinone reduction and proton pumping. Mitochondrial proton-translocating NADH:ubiquinone oxidoreductase (complex I) couples the transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the mitochondrial inner membrane.
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