Solvent isotope effects on interfacial protein electron transfer in crystals and electrode films

D2O-grown crystals of yeast zinc porphyrin substituted cytochrome c peroxidase (ZnCcP) in complex with yeast iso-1-cytochrome c (yCc) diffract to higher resolution (1.7 angstrom) and pack differently than H2O-grown crystals (2.4-3.0 angstrom). Two ZnCcP's bind the same yCc (porphyrin-to-porphyrin separations of 19 and 29 angstrom), with one ZnCcP interacting through the same interface found in the H2O crystals. The triplet excited-state of at least one of the two unique ZnCcP's is quenched by electron transfer (ET) to Fe(III)yCc (k(e) = 220 s(-1)). Measurement of thermal recombination ET between Fe(II)yCc and ZnCcP(+) in the D2O-treated crystals has both slow and fast components that differ by 2 orders of magnitude (k(eb)(1) = 2200 s-1, k(eb)(2) = 30 s(-1)). Back ET in H2O-grown crystals is too fast for observation, but soaking H2O-grown crystals in D2O for hours generates slower back ET, with kinetics similar to those of the D2O-grown crystals (kbl = 7000 s(-1), k(eb)(2) = 100 s(-1)). Protein-film voltammetry of yCc adsorbed to mixed alkanethiol monolayers on gold electrodes shows slower ET for D2O-grown yCc films than for H2O-grown films (k(H) = 800 s(-1); kD = 540 s(-1) at 20 degrees C). Soaking H2O- or D2O-grown films in the counter solvent produces an immediate inverse isotope effect that diminishes over hours until the ET rate reaches that found in the counter solvent. Thus, D2O substitution perturbs interactions and ET between yCc and either CcP or electrode films. The effects derive from slow exchanging protons or solvent molecules that in the crystal produce only small structural changes.