Production and characterisation of Met80X mutants of yeast iso-1-cytochrome c: spectral, photochemical and binding studies on the ferrous derivatives.
The iron ligand, Met80, of yeast iso-1-cytochrome c has been mutated to residues that are unable to bind to the iron. The resultant proteins, Met80Ala, Ser, Asp, Glu, have been expressed and purified. All mutant proteins exhibit well defined pH dependent spectral transitions that report the binding, at high pH, of an intrinsic ligand (probably the nitrogen of an epsilon-NH(2) of a lysine) that drives the heme low-spin. The pK values are mutant dependent. All the mutant proteins bind extrinsic ligands, such as CO, in their ferrous states and we report the apparent quantum yield (phi) for CO photo-dissociation. The values of phi range from 0.004 for Met80Ala to 0.04 for Met80Asp. We also report values for the rate constant for binding the intrinsic lysine residue. The values for this constant, for phi and for the pK values are discussed in terms of the rigidity of the cytochrome structure. We also show that the mutant proteins bind with high affinity to cytochrome c oxidase, both in the ferric and ferrous states. The potential of these proteins to act as light activated electron donors for the study of electron transfer is discussed.
Silkstone
Gary
G
Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK.
Stanway
Glyn
G
Brzezinski
Peter
P
Wilson
Michael T
MT
D000818:Animals; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007834:Lasers; D008024:Ligands; D016296:Mutagenesis / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D010782:Photolysis; D011485:Protein Binding; D011487:Protein Conformation; D011789:Quantum Theory; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013053:Spectrophotometry
D005296:Ferrous Compounds; D008024:Ligands; D002248:Carbon Monoxide; D010088:Oxidoreductases