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

Cytochrome c from the methylotrophic yeast Hansenula polymorpha was isolate d and purified to homogeneity for the first time. The final yield of the hi ghly purified protein from 1.4 kg (wet weight) cells was about 20 mg. The h emoprotein has an apparent molecular mass of 12 kDa and isoelectric point ( pI) of 9.3. The purified protein was characterized by electronic, EPR and N MR spectroscopies. The redox potential of the cytochrome, E degrees, measur ed by cyclic voltammetry measurements at neutral pH, is 0.302 V. Both NMR s pectroscopy and electrochemical measurements confirm the presence in the so lution of several acid-base equilibria, the most pronounced being character ized by a pK(a) of 8.3. The latter pK(a) was attributed to the detachment o f 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. (C) 2000 Elsevier Science B.V. All rights reserved.