| In this work, a flash-induced protein-protein electron transfer (ET) photocycle between cytochrome c (Cc) and cytochrome c peroxidase (CcP) was studied in detail. Photo-triggered redox chemistry from Zn-porphyrins substituted at heme centers previously revealed that Cc:CcP ET reactions depend on solvent conditions, conformational gating, and multiple association sites of varied reactivity. To directly associate ET rates with defined structures, new photochemical methods are developed and applied for studying ET in single protein crystals, where structure can be precisely defined by X-ray crystallography. Measurements of ET rates from Zn-porphyrin-substituted CcP (ZnCcP) to either yeast Cc (yCc) or horse heart Cc (hCc) in crystals indicate that the molecular associations found in the respective crystal structures determine solution reactivity. Similar forward rates for yCc and hCc, despite different orientations relative to CcP, suggest small-amplitude conformational gating of ET even in the crystalline state; faster back ET in the yCc compared to the hCc complex agrees with the relative coupling between redox sites predicted by the structures.; Conservative mutations at the interface can drastically alter the association modes of the partners, emphasizing the sensitivity of protein-protein interactions to subtle structural changes in the interface. Moreover, even minor changes to the interface structure reduce ET recombination reactions relative to the wild-type complex. According to the rate measurements, different association modes generate ET rates that do not correlate with predictions based on cofactor separations or simple bonding pathways. Inhibition of photo-induced ET below 273 K indicates gating by small-amplitude dynamics, even within the crystal. Thus, different associations achieve states of similar reactivity, and within those states conformational fluctuations enable inter-protein ET.; Crystallization of the complex from buffers and proteins exchanged in D2O also results in a new binding mode. Consequently, substitution of D2O for H2O within the crystalline lattice changes the ET properties of the ZnCcP:yCc complex. Structural characterization by X-ray diffraction and solution electrochemistry measurements implicate that the involvement of slowly exchanging protons at the interfacial side-chains and solvent medium interrupt the natural reactivity of the ZnCcP:Cc complex. Therefore, they highlight the sensitivity of ET to subtle structural perturbations. |
