Abstract
Respiratory chain complex I contains 8-9 iron-sulfur clusters. In several cases, the assignment of these clusters to subunits and binding motifs is still ambiguous. To test the proposed ligation of the tetranuclear iron-sulfur cluster N5 of respiratory chain complex I, we replaced the conserved histidine 129 in the 75-kDa subunit from Yarrowia lipolytica with alanine. In the mutant strain, reduced amounts of fully assembled but destabilized complex I could be detected. Deamino-NADH: ubiquinone oxidoreductase activity was abolished completely by the mutation. However, EPR spectroscopic analysis of mutant complex I exhibited an unchanged cluster N5 signal, excluding histidine 129 as a cluster N5 ligand.
Highlights
Little information is available on the spatial arrangement of the other components of the electron transfer pathway
Electron transfer from NADH to the artificial acceptor HAR [23] was only slightly reduced in mitochondrial membranes from strain nuam⌬, pNUAM-H129A and still amounted to 74% of the parental strain value (Table I). This result could be explained by the presence of subcomplexes in the membranes from this mutant; because FMN bound to the 51-kDa subunit is sufficient to catalyze the non-physiological reaction, subcomplexes containing this subunit can show NADH:HAR activity
It should be noted that significant residual NADH:HAR activity was found in the nuam⌬ strain carrying the empty plasmid pUB4 (Table I), no assembled complex I was detectable by BN-PAGE in this strain
Summary
Little information is available on the spatial arrangement of the other components of the electron transfer pathway. Two EPR-silent [Fe4S4] clusters, termed N6a and N6b, have been assigned to the ferredoxin-like subunit of Neurospora crassa complex I homologous to bovine TYKY [10]. The N-terminal part of the 75-kDa subunit contains three highly conserved iron-sulfur cluster motifs and is homologous to the N-terminal portion of iron-only hydrogenases [15]. The fast relaxing [Fe4S4] cluster N5 has been assigned to the second binding motif of the 75-kDa subunit, an unusual HXXXCXXCXXXXXC motif that contains three cysteines and a histidine. To test the proposed assignment of this sequence as a cluster N5 ligation motif in fully assembled complex I, we mutated the conserved histidine 129 of the Y. lipolytica 75-kDa subunit to alanine
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