Modulating radical propagation in proteins by proton-coupled electron transfer and hydrogen bonding

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^•+ radical to increase ET from cytochrome c (Cc) to the active center. W191 substitution to Tyr reduces ET rates, but introduction of an adjacent general base at position 232 (as Glu or His) recovers activity. E232 fluorination shifts the ET pH dependence to lower values, verifying that a hydrogen bond elevates the Y191^• formal potential for effective ET. Photoinitiated ET between Zn-porphyrin (ZnP) CcP (ZnCcP) and Cc also depends on activating Y191 with a basic residue, but through a different mechanism than for the peroxide-driven system. In ZnCcP, pH dependencies and solvent isotope effects indicate that proton-coupled electron transfer to the basic residue and ZnP^•+, respectively, facilitate Y191^• formation. Replacing Cc with the irreversible oxidant [Co(NH3)₅Cl]²⁺ isolates distinct protein radicals for characterization by Electron Paramagnetic Resonance (EPR) spectroscopy. Radical distributions reveal that W191^•+ lies ~15 mV in potential below ZnP^•+ 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^•+/Trp^•+ and the importance of a hydrogen bond to Y191^• 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.