About the enzymes

The proton-translocating NADH-quinone oxidoreductase (complex I) of mammalian mitochondria is composed of 46 unlike subunits and probably has the most intricate structure of any known membrane-associated enzyme complexes. Complex I contains one FMN and at least 8 iron-sulfur clusters as cofactors.

In contrast, the proton-translocating NADH-quinone oxidoreductases (NDH-1) of bacterial respiratory chains is composed of 14 unlike subunits, suggesting that the structure of bacterial NDH-1 is relatively simpler than that of its mitochondrial counterpart. Therefore, bacterial NDH-1 are useful model systems for studying the structure and function of complex I.

Genes encoding NADH-quinone oxidoreductases

In this laboratory, gene clusters encoding Paracoccus and Thermus NDH-1 were cloned and sequenced. These clusters are composed of 14 structural genes (designated NQO1 to NQO14). In addition, DNA sequence of the E. coli NDH-1 operon was reported by Düsseldorf's group.

Expression of individual subunits, assignment of iron-sulfur clusters, architechture of the NDH-1

The NDH-1 and complex I contain at least 5 iron-sulfur clusters. We have expressed individual subunits of NDH-1 and carried out EPR studies to assign the iron-sulfur centers.
 

It is generally accepted that mitochondrial complex I and bacterial NDH-1 can be divided into two sectors, the peripheral segment and the membrane segment. The peripheral segment in the bacterial NDH-1 consists of 7 subunits, including the NQO1-6 and NQO9 subunits. Various expression and extraction studies reported previously suggest that the NQO6 and NQO9 subunits appear to act as connectors between the peripheral and the membrane segments. In addition, these studies also exhibits that subunit NQO1, 2, 3, 6 and 9 house iron-sulfur clusters. In other words, available data indicate that all cofactors of the bacterial NDH-1 are located in the peripheral subunits.

Membrane segment of NDH-1

The membrane-associated segment of the bacterial NDH-1 appears to be composed of 7 subunits (NQO7,8, and 10 - 14). However, in contrast to the peripheral segment, this membrane associated segment is believed to be involved in the proton translocation of the bacterial NDH-1. Therefore, understanding the properties and characteristics of the NQO7, 8, and 10 - 14 subunits is a prerequisite to understanding the mechanism of proton translocation of the bacterial NDH-1. To date, little is known about structural properties of these subunits. In addition, there has not been any report of successful expression of these hydrophobic subunits which would make studies of their biochemical and physicochemical properties possible. Therefore, we first attempted to express the Paracoccus NQO7 subunit (a counterpart of mitochondrial ND3 subunit) in E. coli in a hope that similar expression strategy may be useful for structural studies of other hydrophobic subunits.

The expression of the NQO7 subunit was achieved using GST fusion system. The GST-fused NQO7 subunit was expressed in the membrane fraction of the host cells and was extracted from the membranes by detergents. The extracted polypeptide was purified and characterized. Utilizing immunochemical and cysteine residue modification technique, the topology of the Paracoccus NQO7, NQO10 and NQO11 subunits in the membrane has been determined. A group in Sweden reported the topology of NQO8 (ND1) and NQO12 (ND5).