Reaction rates extracted from measurements of donor luminescence quenching by randomly dispersed electron acceptors reveal an exponential decay constant of 1.23 per angstrom for electron tunneling through a frozen toluene glass (with a barrier to tunneling of 1.4 electron volts). The decay constant is 1.62 per angstrom (the barrier, 2.6 electron volts) in a frozen 2-methyl-tetrahydrofuran glass. Comparison to decay constants for tunneling across covalently linked xylyl (0.76 per angstrom) and alkyl (1.0 per angstrom) bridges leads to the conclusion that tunneling between solvent molecules separated by approximately 2 angstroms (van der Waals contact) is 20 to 50 times slower than tunneling through a comparable length of a covalently bonded bridge. Our results provide experimental confirmation that covalently bonded pathways can facilitate electron flow through folded polypeptide structures.

Well-defined voltammetric responses of redox proteins with acidic-to-neutral pI values have been obtained on pure alkanethiol as well as on mixed self-assembled-monolayer (SAM) omega-derivatized alkanethiol/gold bead electrodes. Both azurin (P. aeruginosa) (pI = 5.6) and subunit II (Cu(A) domain) of ba(3)-type cytochrome c oxidase (T. thermophilus) (pI = 6.0) exhibit optimal voltammetric responses on 1:1 mixtures of [H(3)C(CH(2))(n)()SH + HO(CH(2))(n)()SH] SAMs. The electron transfer (ET) rate vs distance behavior of azurin and Cu(A) is independent of the omega-derivatized alkanethiol SAM headgroups. Strikingly, only wild-type azurin and mutants containing Trp48 give voltammetric responses: based on modeling, we suggest that electronic coupling with the SAM headgroup (H(3)C- and/or HO-) occurs at the Asn47 side chain carbonyl oxygen and that an Asn47-Cys112 hydrogen bond promotes intramolecular ET to the copper. Inspection of models also indicates that the Cu(A) domain of ba(3)-type cytochrome c oxidase is coupled to the SAM headgroup (H(3)C- and/or HO-) near the main chain carbonyl oxygen of Cys153 and that Phe88 (analogous to Trp143 in subunit II of cytochrome c oxidase from R. sphaeroides) is not involved in the dominant tunneling pathway. Our work suggests that hydrogen bonds from hydroxyl or other proton-donor groups to carbonyl oxygens potentially can facilitate intermolecular ET between physiological redox partners.

Tryptophan radicals play a significant role in mediating biological electron transfer. We report the photogeneration of a long-lived, neutral tryptophan radical (Az48W*) from the native residue tryptophan-48 in the hydrophobic core of azurin. The optical absorption, electron paramagnetic resonance, and resonance Raman spectra strongly support the formation of a neutral radical, and the data are consistent with direct electron transfer between tryptophan and the copper(II) center. Spectra of the long-lived Az48W* species are compared to those of a previously studied, solvent-exposed radical at position 108 to identify signatures of tryptophan radicals that are sensitive to the local environment. The absorption maxima of Az48W* display an approximately 23 nm hypsochromic shift in the nonpolar environment. The majority of the resonance Raman frequencies are downshifted by approximately 7 cm(-1) relative to the solvent-exposed radical, and large changes in intensity are observed for some modes. The resonance Raman excitation profiles for Az48W* exhibit distinct maxima within the absorption envelope. Electron paramagnetic resonance spectroscopy yields spectra with partially resolved lines caused by hyperfine couplings; the differences between the coupling constants for the buried and solvent-exposed radical are primarily caused by variations in structure. The insights gained by electronic, vibrational, and magnetic resonance spectroscopy enhance our fundamental understanding of the effects of protein environment on radical properties. Hypotheses for the proton transfer pathway within azurin and a deprotonation rate of approximately 5 x 10(6) s(-1) are proposed.

The triplet metal-to-ligand charge transfer ((3)MLCT) dynamics of two structurally characterized Re(I)(CO)(3)(phen)(HisX)-modified (phen = 1,10-phenanthroline; X = 83, 109) Pseudomonas aeruginosa azurins have been investigated by picosecond time-resolved infrared (TRIR) spectroscopy in aqueous (D(2)O) solution. The (3)MLCT relaxation dynamics exhibited by the two Re(I)-azurins are very different from those of the sensitizer [Re(I)(CO)(3)(phen)(im)](+) (im = imidazole). Whereas the Re(I)(CO)(3) intramolecular vibrational relaxation in Re(I)(CO)(3)(phen)(HisX)Az (4 ps) is similar to that of [Re(I)(CO)(3)(phen)(im)](+) (2 ps), the medium relaxation is much slower ( approximately 250 vs 9.5 ps); the 250-ps relaxation is attributable to reorientation of D(2)O molecules as well as structural reorganization of the rhenium chromophore and nearby polar amino acids in each of the modified proteins.