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.

Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria

“…Complex I was prepared from Bos taurus (bovine) heart mitochondria [20], Pichia pastoris [21] and Escherichia coli [22], as described previously. SMPs (submitochondrial particles) and mitochondrial membranes were prepared from bovine heart mitochondria [20,23].…”

The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

“…For some purified eukaryotic, non-mammalian enzymes the Fe:FMN ratio's are 27.5 (Torulopsis utilis (Tottmar and Ragan 1971)), 28-38 (N. crassa (Schulte et al 1998)), 33.2 (Pichia pastoris (Bridges et al 2009)) and 30.4 (Pichia angusta (Bridges et al 2009)). The values for the purified Y. lipolytica enzyme have not been reported (Djafarzadeh et al 2000;Kashani-Poor et al 2001a).…”

Exploiting mitochondria as targets for the development of new antifungals

“…NUZM is fungal-specific. [13][14][15][16][17]24,25 From the Candida Genome Database, we have identified NUXM as orf19.6607 (NUO1) and NUZM as orf19.287 (NUO2). We have shown that both proteins are NADH: Ubiquinone Oxidoreductases.…”

“…These data have been rather easy to obtain, given the extensive data bases that allow comparisons across species including human mitochondria. [13][14][15][16][17] If a search focuses upon new targets for antifungal drug discovery, specificity should be an initial descriptor followed by the verification of functions of the selected gene. The latter requires mutation/deletion of the encoding gene to profile dysfunction(s), and by inference, direct or indirect functions.…”