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^dot+ 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 (as Glu or His) at position 232 (Y191:E/H232 CcP) recovers activity. E232 fluorination lowers the pKa of the conjugate base and confirms that a hydrogen bond is critical to elevate the Y191^dot 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^dot+, respectively, facilitates Y191^dot 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 and computation indicate that W191^dot+ lies close in potential to ZnP^dot+ and that the two radicals exchange on a slow time scale despite their close separation. Remarkably, Y191:E/H232 ZnCcP variants propagate radicals differently to peripheral sites depending on the nature of the 232 residue. QM/MM calculations support radical exchange between ZnP^dot+/Trp^dot+ and the importance of a hydrogen bond to Y191^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.