Cytochrome c Interaction with Yeast Cytochrome b2

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Abstract
Fluorescent derivatives of cytochrome c were prepared by replacing the heme iron with closed-shell metals such as zinc or tin. The iron-free derivatives of cytochrome c bind to yeast lactate dehydrogenase (cytochrome b2) stoichiometrically and with high affinity. Spectral overlap exists between the fluorescence of porphyrin, Zn(II) or Sn(IV) cytochrome c and the absorption of the heme of cytochrome b; therefore dipole-dipole interaction is possible as predicted by Forster's theory of energy transfer. Changes in the fluorescence yield and the fluorescent decay profile of the cytochrome c derivatives are consistent with the view that the heme distance is sufficiently close for dipolar interactions. The distance calculated from the data depends upon assumptions in the theory for energy transfer and uncertainties in the experiment. It can be argued that due to the symmetry of the metalloporphyrins the relative orientations of the two hemes do not introduce a significant uncertainty in the calculation. However the decay profiles of the iron-free cytochromes are complex, possibly reflecting structural rearrangement of the polypeptide chain during the fluorescent lifetime. The steady-state fluorescent yields would indicate that the mean distance is around 1.8 nm