A reductase/isomerase subunit of mitochondrial NADH : ubiquinone oxidoreductase (complex I) carries an NADPH and is involved in the biogenesis of thecomplex

Respiratory chains of bacteria and mitochondria contain closely related for ms of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I. The bacterial complex I consists of 14 subunits, whereas the mitochondrial comp lex contains some 25 extra subunits in addition to the homologues of the ba cterial subunits. One of these extra subunits with a molecular mass of 40 k Da belongs to a heterogeneous family of reductases/isomerases with a conser ved nucleotide binding site. We deleted this subunit in Neurospora crassa b y gene disruption. In the mutant nuo40, a complex I lacking the 40 kDa subu nit is assembled. The mutant complex I does not contain tightly bound NADPH present in wild-type complex I. This NADPH cofactor is not connected to th e respiratory electron pathway of complex I. The mutant complex has normal NADH dehydrogenase activity and contains the redox groups known for wild-ty pe complex I, one flavin mononucleotide and four iron-sulfur clusters detec table by electron paramagnetic resonance spectroscopy. In the mutant comple x these groups are all readily reduced by NADH. However, the mutant complex is not capable of reducing ubiquinone. A recently described redox group id entified in wild-type complex I by UV-visible spectroscopy is not detectabl e in the mutant complex. We propose that the reductase/isomerase subunit wi th its NADPH cofactor takes part in the biosynthesis of this new redox grou p. (C) 1999 Academic Press.