The respiratory complexes I from the mitochondria of two Pichia species

Abstract

NADH:ubiquinone oxidoreductase (complex I) is an entry point for electrons into the respiratory chain in many eukaryotes. It couples NADH oxidation and ubiquinone reduction to proton translocation across the mitochondrial inner membrane. Because complex I deficiencies occur in a wide range of neuromuscular diseases, including Parkinson's disease, there is a clear need for model eukaryotic systems to facilitate structural, functional and mutational studies. In the present study, we describe the purification and characterization of the complexes I from two yeast species, Pichia pastoris and Pichia angusta. They are obligate aerobes which grow to very high cell densities on simple medium, as yeast-like, spheroidal cells. Both Pichia enzymes catalyse inhibitor-sensitive NADH:ubiquinone oxidoreduction, display EPR spectra which match closely to those from other eukaryotic complexes I, and show patterns characteristic of complex I in SDS/PAGE analysis. Mass spectrometry was used to identify several canonical complex I subunits. Purified P. pastoris complex I has a particularly high specific activity, and incorporating it into liposomes demonstrates that NADH oxidation is coupled to the generation of a protonmotive force. Interestingly, the rate of NADH-induced superoxide production by the Pichia enzymes is more than twice as high as that of the Bos taurus enzyme. Our results both resolve previous disagreement about whether Pichia species encode complex I, furthering understanding of the evolution of complex I within dikarya, and they provide two new, robust and highly active model systems for study of the structure and catalytic mechanism of eukaryotic complexes I.