Isolation and physico-chemical characterization of a cytochrome c from the methylotrophic yeast Hansenula polymorpha

Cytochrome c from the methylotrophic yeast Hansenula polymorpha was isolated and purified to homogeneity for the first time. The final yield of the highly purified protein from 1.4 kg (wet weight) cells was about 20 mg. The hemoprotein has an apparent molecular mass of 12 kDa and isoelectric point (pI) of 9.3. The purified protein was characterized by electronic, EPR and NMR spectroscopies. The redox potential of the cytochrome, E degrees, measured by cyclic voltammetry measurements at neutral pH, is 0.302 V. Both NMR spectroscopy and electrochemical measurements confirm the presence in the solution of several acid-base equilibria, the most pronounced being characterized by a pK(a) of 8.3. The latter pK(a) was attributed to the detachment of the iron(III) ion-coordinated methionine and its replacement by a lysine residue. The electrochemically derived thermodynamic parameters for neutral and alkaline protein species (DeltaS degrees (rc) and DeltaH degrees (rc)) were obtained from the temperature dependence of the redox potential.     

Study of the Hansenula anomala yeast flavocytochrome-b2--cytochrome-c complex 1. Characterization of fluorescent Zn(II)-substituted cytochrome c

Substitution of Fe2+ for the Zn2+ ion in Hansenula anomala cytochrome c provides a luminescent derivative suitable as a probe for the determination of the interaction of cytochrome c with H. anomala flavocytochrome b2; its light absorption and fluorescence properties have been characterized. H. anomala Zn-cytochrome c appears to be in the form of a stable though non-covalent dimer from molecular weight determinations performed using gel filtration, polyacrylamide gel electrophoresis under denaturing conditions, and ultracentrifugation methods. By contrast, metal-free porphyrin-cytochrome c, the precursor of Zn-cytochrome c obtained upon removal of iron from cytochrome c in cold anhydrous fluorhydric acid, had the same partition coefficient as native cytochrome c through conventional gel filtration. Significant conformational perturbations of H. anomala cytochrome c should therefore follow from Zn2+ incorporation into the porphyrin c moiety. Titrations at low ionic strength with native, tetrameric H. anomala flavocytochrome b2 in the lactate-reduced state showed a simple binding equilibrium (Kd = 0.1 microM at I = 0.03 M, 10 degrees C) with a stoichiometry of one Zn-cytochrome c dimer per protomer of flavocytochrome b2. Quenching of the Zn-porphyrin c fluorescence within this complex was much larger (43%) than reported by other authors using cytochrome c and flavocytochrome b2 from different sources.     

Proteolysis of L-(+)-lactate cytochrome c oxidoreductase (cytochrome b2) extracted from Saccharomyces cerevisiae and Hansenula anomala yeasts.

The L-(+)-Lactate:cytochrome c oxidoreductase or cytochrome b2 from the yeasts Saccharomyces cerevisiae and Hansenula anomala were partially hydrolysed in various concentrations of trypsin. Conditions were found which allowed the isolation from the Hansenula enzyme of a 140 000 +/- 10 000-dalton flavoprotein. The prosthetic flavin groups were still reducible by substrate (spectroscopic evidence) but the flavoprotein was unable to form a complex with cytochrome c, the physiological acceptor in the enzymatic reaction. No such flavoprotein units could be found during proteolysis of the Saccharomyces enzyme. The heme prosthetic group of the Hansenula enzyme remained bound to a 15 500 +/- 1000-dalton protein unit which was larger than, but very similar to, the well known 'cytochrome b2 core' of the Saccharomyces enzyme. Moreover, the degradation of different enzyme samples by contaminated proteases allowed the isolation of a particular form of Hansenula enzyme: each tetramer had, on the mean, four bound flavins and only two heme groups. These molecules completely retained their ability to form a complex with cytochrome c.     

Deacidification of grape juice with derepressed mutants of the yeast Hansenula anomala

Summary Transport and utilization of malic acid by the yeast Hansenula anomala are subject to glucose repression. Derepressed diploid mutant strains were obtained by hybridization of derepressed haploid mutant strains of opposite mating type. Six diploid mutant strains displayed derepressed behaviour with respect to malic acid utilization in the presence of glucose up to 30% (w/v). Three of these diploid mutant strains, as compared with the parent strain, were able to degrade completely malic acid in grape juice without fermenting the sugars. In addition, using one diploid mutant strain together with a strain of the wine yeast Saccharomyces cerevisiae, it was possible to carry out a mixedmicrovinification in which deacidification occurred simultaneously with alcoholic fermentation.     

Fluorescence studies on the interaction between two cytochromes extracted from the yeast, Hansenula anomala

The binding of cytochrome c to the cytochrome b2 core, both extracted from the yeast, Hansenula anomala, has been studied. Cytochrome b2 core heme is extracted and replaced by the fluorescent probe, 2-p-toluidinylnaphthalene-6-sulfonate (TNS). A dissociation constant in the range of 85 microM is found for the TNS-apoprotein complex with a stoichiometry of 1:1. The interaction between the two proteins is followed by monitoring changes in the TNS fluorescence. We find the interaction between the cytochrome c and the apocytochrome b2 core to be dependent upon the ionic strength. The dissociation constant of this complex at 20 mM ionic strength is 6 +/- 2 microM with a 1:1 stoichiometry. This dissociation constant is similar to that estimated, by other researchers, for the dimer Zn cytochrome c-cytochrome b2 core complex.     

Development of an integrative DNA transformation system for the yeast Hansenula anomala

A host-vector system for the yeast Hansenula anomala was developed. The system was based on an auxotrophic mutant host of H. anomala which was defective in orotidine-5′-phosphate decarboxylase (ODCase) activity. The H. anomala ODCase-negative mutant strains (ura3 strains) were isolated based on 5-fluoroorotic acid (5-FOA) resistance. A plasmid vector containing the H. anomala URA3 gene was used for transformation. Using this plasmid, all of the H. anomala ura3 strains tested could be transformed to Ura⁺ phenotypes. In all of Ura⁺ transformants, the introduced plasmid was integrated into the chromosomal URA3 locus by homologous recombination. The Ura⁺ phenotype of the transformants was stably maintained after nonselective growth.     

Solution NMR study of the yeast cytochrome c peroxidase: cytochrome c interaction

Here we present a solution NMR study of the complex between yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP), a paradigm for understanding the biological electron transfer. Performed for the first time, the CcP-observed heteronuclear NMR experiments were used to probe the Cc binding in solution. Combining the Cc- and CcP-detected experiments, the binding interface on both proteins was mapped out, confirming that the X-ray structure of the complex is maintained in solution. Using NMR titrations and chemical shift perturbation analysis, we show that the interaction is independent of the CcP spin-state and is only weakly affected by the Cc redox state. Based on these findings, we argue that the complex of the ferrous Cc and the cyanide-bound CcP is a good mimic of the catalytically-active Cc–CcP compound I species. Finally, no chemical shift perturbations due to the Cc binding at the low-affinity CcP site were observed at low ionic strength. We discuss possible reasons for the absence of the effects and outline future research directions.     

Transport of malic acid and other dicarboxylic acids in the yeast Hansenula anomala

DL-Malic acid-grown cells of the yeast Hansenula anomala formed a saturable transport system that mediated accumulative transport of L-malic acid with the following kinetic parameters at pH 5.0: Vmax, 0.20 nmol.s-1.mg (dry weight)-1; Km, 0.076 mM L-malate. Uptake of malic acid was accompanied by proton disappearance from the external medium with rates that followed Michaelis-Menten kinetics as a function of malic acid concentration. Fumaric acid, alpha-ketoglutaric acid, oxaloacetic acid, D-malic acid, and L-malic acid were competitive inhibitors of succinic acid transport, and all induced proton movements that followed Michaelis-Menten kinetics, suggesting that all of these dicarboxylates used the same transport system. Maleic acid, malonic acid, oxalic acid, and L-(+)-tartaric acid, as well as other Krebs cycle acids such as citric and isocitric acids, were not accepted by the malate transport system. Km measurements as a function of pH suggested that the anionic forms of the acids were transported by an accumulative dicarboxylate proton symporter. The accumulation ratio at pH 5.0 was about 40. The malate system was inducible and was subject to glucose repression. Undissociated succinic acid entered the cells slowly by simple diffusion. The permeability of the cells by undissociated acid increased with pH, with the diffusion constant increasing 100-fold between pH 3.0 and 6.0.     

Treatment and phosphorus removal from high-concentration organic wastewater by the yeast Hansenula anomala J224 PAWA

A flocculent yeast, Hansenula anomala J224 PAWA, bred in this study, accumulated twice as much phosphorus as the wild type. Over a 30-d period, PAWA removed 70–80% of dissolved total phosphorus from sweet-potato and barley shochu wastewaters (alcoholic distillery wastewaters) while the wild type removed only 30%. Waste sludge was easily separated from effluent wastewater because PAWA cells made large flocks that rapidly settled. Component analysis suggested that PAWA sludge could be used as a protein source for feedstuff and as a phosphorus source for fertilizer. Under anaerobic conditions, denitrification was rapid, resulting in the removal of large amounts of nitrogen from barley shochu wastewater. These results suggest that small shochu manufacturers could benefit from using PAWA to remove phosphorus and organic compounds and then by using a combination of the upflow anaerobic sludge blanket and the downflow hanging sponge method (UASB-DHS method) for nitrification/denitrification.     

Transport of malic acid and other dicarboxylic acids in the yeast Hansenula anomala.

DL-Malic acid-grown cells of the yeast Hansenula anomala formed a saturable transport system that mediated accumulative transport of L-malic acid with the following kinetic parameters at pH 5.0: Vmax, 0.20 nmol.s-1.mg (dry weight)-1; Km, 0.076 mM L-malate. Uptake of malic acid was accompanied by proton disappearance from the external medium with rates that followed Michaelis-Menten kinetics as a function of malic acid concentration. Fumaric acid, alpha-ketoglutaric acid, oxaloacetic acid, D-malic acid, and L-malic acid were competitive inhibitors of succinic acid transport, and all induced proton movements that followed Michaelis-Menten kinetics, suggesting that all of these dicarboxylates used the same transport system. Maleic acid, malonic acid, oxalic acid, and L-(+)-tartaric acid, as well as other Krebs cycle acids such as citric and isocitric acids, were not accepted by the malate transport system. Km measurements as a function of pH suggested that the anionic forms of the acids were transported by an accumulative dicarboxylate proton symporter. The accumulation ratio at pH 5.0 was about 40. The malate system was inducible and was subject to glucose repression. Undissociated succinic acid entered the cells slowly by simple diffusion. The permeability of the cells by undissociated acid increased with pH, with the diffusion constant increasing 100-fold between pH 3.0 and 6.0.     

Hydroxyl radical oxidation of cytochrome c by aerobic radiolysis

The reaction of radiolytically generated *OH with cytochrome c was investigated by mass spectrometry. Tryptic digestion and characterization of the oxidized peptides by MALDI-TOF and ESI tandem mass spectrometry identified eight different amino acid residues with oxidized side chains with no cleavage of the protein detected. Solvent-accessible aromatic and methionine residues are the most susceptible to oxidation by *OH. These results support the careful use of *OH in characterizing protein surfaces. Dose-response studies identified the residues most prone to oxidation to be Phe-36, Phe-46, and Met-80. Hydroxylation of Phe-36 and Phe-46 should serve as indicators of the presence of *OH in the mitochondrial intermembrane space. Using solutions containing 50 at.% (18)O, our study also provides a novel method of determining the source of oxygen during *OH-mediated oxidation of proteins and contributes to identification of the modified residue type, with Phe>Tyr>Met in (18)O incorporation. During aerobic radiolysis, UV-vis spectroscopy indicates that ferrocytochrome c reaches a steady state concomitant with reduction of the heme.     

Influence of oxygen on the synthesis of flavocytochrome b2 by the yeast Hansenula anomala

Summary Control of oxygen concentration in the culture medium during growth of the yeast Hansenula anomala on l-lactate as sole carbon source allows induction of the synthesis of flavocytochrome b2 or l-lactate cytochrome-c oxydoreductase (E.C. 1.1.2.3.). This phenomenon is accompanied by an important change in the yeast doubling time.     

Pathogenicity of Hansenula anomala in a model of immunocompromised mice

Systemic infections caused by opportunistic fungi have shown an increased frequency in the past 10 years, particularly in immunocompromised patients. Hansenula anomala is an ascosporogenous yeast of the Ascomycetes class found in the skin, throat, and digestive tract transient normal flora. This study was conducted to compare the pathogenicity of H. anomala and Candida albicans in a model of immunocompromised mice. Thirty-eight Swiss mice were divided into two groups as follows: 30 animals received an intraperitoneal (i.p.) injection of cyclophosphamide (200 mg/kg) four days before the induction of infection with H. anomala (1 × 10⁶ yeasts/mL), and 8 animals received 100 mg/kg of cyclophosphamide at 3-day intervals during 3 weeks before inoculation of 1 × 10⁷ yeasts/mL. All animals were treated with amoxicillin/clavulanic acid (40 mg/kg) four days before induction of infection. A group of mice inoculatd with C. albicans (ATCC 64548) served as control. Tissue samples from the lung, spleen, liver, and kidney for histological and mycologic studies were obtained at necropsy. In each animal, the number of viable yeasts per gram of kidney was determined. The organs most frequently infected by H. anomala were the kidneys and the liver (20%), and the lung (10%). However, in conditions of sustained immunosuppression, H. anomala was found in 65.5% of the organs examined. It is concluded that in an experimental model of immunocompromised mice, the pathogenicity of H. anomala was low. This revised version was published online in August 2006 with corrections to the Cover Date.