Structural change of plastocyanin (PC) due to the interaction with lysine peptides (Lysptd's) has been studied by absorption, resonance Raman, and electrochemical measurements and by measuring the electron transfer between PC and cytochrome c (cyt c) in the presence of Lysptd. Absorption spectral changes which were observed when Lysptd's up to penta-lysine were added to PC solution have been ascribed by resonance Raman studies to the change in the active site Cu−cysteine geometry upon binding of Lysptd to the PC negative patch. The same spectral changes were observed for the PC−cyt c interaction. Electrochemical measurements showed that the redox potential of PC increases upon Lysptd binding, suggesting that Lysptd's induce a structural change in PC through the copper ligating cysteine residue to make the copper site adapted for facile electron transfer. Lysptd's competitively inhibited the electron transfer from reduced cyt c to oxidized PC, which indicated that they function as models of the PC interacting site of proteins. The effects of Lysptd on electron transfer are explained as competitive inhibition due to neutralization of the PC negative patch by formation of PC·Lysptd complexes. The electron-transfer rate from reduced cyt c to oxidized PC and the inhibiting effect of Lysptd decreased upon decreasing the net charge of the negative patch by mutation. The structural change of PC was also found to decrease significantly with these mutants. The present observations strongly support that the PC negative patch is the dominant cyt c/f molecular recognition site and open up the possibility that charged peptides can be used for studying protein−protein interactions in a systematic way.

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Cytochrome c (cyt c) and cytochrome f (cyt f) molecular recognition characters and their structural changes due to complex formation with negatively charged aspartic acid peptides (Aspptd's) have been studied. Changes in the absorption spectrum of cyt c in the Soret region were detected when Aspptd's, up to penta-Asp, were added to the cyt c solution. These changes were the same as those observed when cyt c interacted with plastocyanin (PC), indicating that Aspptd's interacted with cyt c in the same way as PC. Conformational changes of cyt c due to interaction with Aspptd's observed by resonance Raman spectroscopy were similar to those reported for cyt c when bound with its native partner, cytochrome c oxidase. Electrochemical measurements showed that the redox potential of cyt c and cyt f shifted to lower potentials by 7−20 mV upon Aspptd binding, showing the enhancement in the electron donor ability of both cyt c and cyt f upon complex formation with Aspptd. The changes in the absorption spectrum and redox potential increased with the length and concentration of Aspptd. The observed structural and redox changes of cyt c and cyt f are attributed to adduct formations with Aspptd's by electrostatic interactions and suggest that similar changes would occur for cyt c and cyt f when interacting with proteins. Aspptd's, tetra- and penta-aspartic acid, served as competitive inhibitors of the electron transfer from cyt c or cyt f to PC, which was ascribable to the same adduct formation.