Zawistowski, R. K.; Chauvire, T.; Manna, S.; Ananth, N.; CRANE, B. R.

Long-range protein electron transfer (ET) often depends on tryptophan and tyrosine residues acting as radical relay sites. For example, Cytochrome c peroxidase (CcP) generates a W191{middle dot}+ radical to accelerate ET from cytochrome c (Cc). W191 substitution to Tyr reduces ET rates, but introduction of an adjacent general base at position 232 (Glu/His) recovers activity. E232 fluorination shifts the ET pH dependence to lower values, thereby confirming that a hydrogen bond elevates the Y191{middle dot} formal potential for effective ET. Photoinitiated ET between Zn-porphyrin (ZnP) CcP (ZnCcP) and Cc also depends on coupling a base to Y191, but through a different mechanism than the peroxide-driven system. In ZnCcP, pH dependencies and solvent isotope effects indicate that proton-coupled electron transfer to the adjacent basic residue and ZnP{middle dot}+, respectively, facilitate Y191{middle dot} formation. Replacing Cc with the irreversible oxidant [Co(NH3)5Cl]2+ isolates distinct protein radicals for characterization by continuous-wave Electron Paramagnetic Resonance (EPR) and pulse EPR. Radical distributions reveal that W191{middle dot}+ lies ~15 mV in potential below ZnP{middle dot}+ and that the two radicals exchange on a slow time scale despite their close separation. Remarkably, ZnCcP Y,G191:E,H232 variants propagate radicals differently to peripheral sites depending on the nature of the 232 residue. QM/MM calculations support radical exchange between ZnP{middle dot}+/Trp{middle dot}+ and the importance of a hydrogen bond to Y191{middle dot} for maintaining a high potential to oxidize peripheral donors. These resolved reactivity patterns of CcP/ZnCcP have general relevance for engineering proton management to separate and migrate charge in proteins and potentially other molecular systems.