Production and characterisation of Met80X mutants of yeast iso-1-cytochrome c: spectral, photochemical and binding studies on the ferrous derivatives Original Research Article

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 ε-NH2 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 (ϕ) for CO photo-dissociation. The values of ϕ 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 ϕ 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.