Characterization of redox centers and electron transfer in NADH:quinone oxidoreductase of fungal and bacterial systems
Abstract
To elucidate the reaction mechanism of mammalian complex I (NADH:ubiquinone oxidoreductase EC.1.6.5.3), comparative studies of simpler NADH:quinone oxidoreductases in eucaryotic Neurospora crassa and bacterial Thermus thermophilus HB-8 systems have been conducted. Normal complex I (large complex I) of N. crassa is essentially equivalent to the bovine heart complex I; and can be further resolved into hydrophobic and hydrophilic fractions. EPR and potentiometric characterizations indicate that the iron-sulfur clusters of N. crassa large complex I are similar to the mammalian counterparts. It contains one binuclear and two tetranuclear iron-sulfur clusters, comprising an iso-potential group, and one additional tetranuclear cluster with much higher E$\sb{\rm m}$ value. In the presence of a mitochondrial protein synthase inhibitor, chloramphenicol, N. crassa cells make a truncated form of complex I (small complex I), which is identical to the hydrophilic portion of the large complex I. The small complex I of N. crassa contains three clusters of the iso-potential group and lacks the cluster with high E$\sb{\rm m}$ which localizes in the hydrophobic fraction. Complex I of T. thermophilus HB-8 (NDH-1) consists of about 10 subunits and shows an indication for energy coupling. NDH-1 in T. thermophilus membranes is fully sensitive to the well known inhibitors of complex I in mammalian tissue, namely rotenone and piericidin A. The I$\sb{50}$, however, is much higher in the NDH-1. EPR and potentiometric analysis reveal that the NDH-1 of T. thermophilus HB-8 contains only three iron-sulfur clusters as the iso-potential group, which are not direct proton carriers. Analysis of free radical signals shows that two distinct Q-binding sites belong to NDH-1. One scenario is that quinone and clusters of the iso-potential group together may contribute to energy coupling in NDH-1. Present results raise a possibility that role for FMN as a proton carrier at site I may not be so simple as previously proposed. One simple method for large scale NDH-1 purification is described.
Subject Area
Biophysics|Biology
Recommended Citation
Wang, Da-Cheng, "Characterization of redox centers and electron transfer in NADH:quinone oxidoreductase of fungal and bacterial systems" (1991). Dissertations available from ProQuest. AAI9200400.
https://repository.upenn.edu/dissertations/AAI9200400