Although bonding networks determine electron-transfer (ET) rates within proteins, the mechanism by which structure and dynamics influence ET across protein interfaces is not well understood. Measurements of photochemically induced ET and subsequent charge recombination between Zn-porphyrin-substituted cytochrome c peroxidase and cytochrome c in single crystals correlate reactivity with defined structures for different association modes of the redox partners. Structures and ET rates in crystals are consistent with tryptophan oxidation mediating charge recombination reactions. Conservative mutations at the interface can drastically affect how the proteins orient and dispose redox centers. Whereas some configurations are ET inactive, the wild-type complex exhibits the fastest recombination rate. Other association modes generate ET rates that do not correlate with predictions based on cofactor separations or simple bonding pathways. Inhibition of photoinduced ET at <273 K indicates gating by smallamplitude dynamics, even within the crystal. Thus, different associations achieve states of similar reactivity, and within those states conformational fluctuations enable interprotein ET.cytochrome Í protein dynamics Í protein-protein interaction Í electron tunneling M any long-range electron-transfer (ET) reactions in biology occur across transient protein-protein interfaces. Reaction rates depend on factors that control both electron tunneling and conformational dynamics coupled to protein association processes (1-3). As such, interprotein ET is sensitive to structure and dynamics at the interface (1, 4-11). Residue substitution (achieved either by use of protein homologs, site-directed mutants, or computations) has been a popular and powerful approach for probing how interface composition influences interprotein ET (7,(10)(11)(12)(13)(14). Nevertheless, effects of residue variation on interface structure are not often known.The natural redox partners yeast cytochrome c peroxidase (CcP) and yeast cytochrome c (yCc), whose structure as a complex was first determined in 1992 (15) and later as a covalent complex (16), have served as a paradigm for studying interprotein ET reactions (8,17). Hoffman and colleagues (8) have exploited ZnCcP substitution to photoactivate ET reactions and examine the effects of many structural and chemical perturbations on interprotein ET. In this system, the ZnCcP triplet state ( 3 ZnCcP) reduces Fe(III)Cc, and then back ET recombines the charge separation (Fig. 1). Recently, it has been demonstrated that a Trp-191 3 Phe CcP variant has much slower ET back-rates (k eb ) than wild-type (WT) CcP in the 1:1 complex with yCc (18). Thus, electron hopping through Trp-191 may accelerate the recombination reaction (Fig. 1), in analogy to the natural reaction between CcP compound I and Fe(II)Cc (19). Nevertheless, much slower ET back-rates in the complex between CcP and hCc compared with yCc, both in solution (20) and in crystals (21), indicate that ET across the protein-protein interface limits the overa...