Kinetic experiments have conclusively shown that electron transfer can take place over large distances (greater than 10 angstroms) through protein interiors. Current research focuses on the elucidation of the factors that determine the rates of long-range electron-transfer reactions in modified proteins and protein complexes. Factors receiving experimental and theoretical attention include the donor-acceptor distance, changes in geometry of the donor and acceptor upon electron transfer, and the thermodynamic driving force. Recent experimental work on heme proteins indicates that the electron-transfer rate falls off exponentially with donor-acceptor distance at long range. The rate is greatly enhanced in proteins in which the structural changes accompanying electron transfer are very small.

Spectroscopic and electrochemical studies, incorporating electronic spectra, electron paramagnetic resonance (EPR) spectra, resonance Raman (RR) spectra, and measurements of the redox potential, have been carried out on the blue copper protein azurin, from Alcaligenes denitrificans. These data are correlated with the refined crystal structure of this azurin and with corresponding data for other blue copper proteins. The electronic spectrum, characterized by an intense (epsilon = 5100 M-1 cm-1) charge-transfer band at 619 nm, the EPR spectral parameters (g perpendicular = 2.059, g parallel of = 2.255, A parallel of = 60 X 10(-4) cm-1), and the resonance Raman spectrum are similar to those obtained from other azurins and from plastocyanins. Both the electronic spectrum and the EPR spectrum are unchanged over the pH range 4-10.5, but major changes occur above pH 12 and below pH 3.5. A small reversible change occurs at pH approximately 11.4. In the RR spectrum the Cu-S stretching mode is shown to contribute to all of the five principal RR peaks. Deuterium substitution produces shifts in at least seven of the peaks; these shifts may be attributable, at least in part, to the NH...S hydrogen bond to the copper-ligated Cys-112. Measurements of the redox potential, using spectroelectrochemical methods, over the temperature range 4.8-40.0 degrees C, give values for delta H0' and delta S0' of -55.6 kJ mol-1 and -97.0 J K-1 mol-1, respectively. The redox potential of A. denitrificans azurin at pH 7.0, Eo', is 276 mV. These data are interpreted in terms of a copper site, in azurin, comprising three strong bonds, in an approximately trigonal plane, from Cys-112, His-46, and His-117 and much longer axial approaches from Met-121 and the peptide carbonyl oxygen of Gly-45. Spectral differences within the azurin family and between azurin and plastocyanin are attributed to differences in the strengths of these axial interactions. Likewise, the distinctly lower Eo values for azurins, as compared with plastocyanins, are related to the more copper(II)-like site in azurin [with a weaker Cu-S(Met) interaction and a Cu-O interaction not found in plastocyanin]. On the other hand, the relative constancy of the EPR parameters between azurin and plastocyanin suggests they are not strongly influenced by weakly interacting axial groups.

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The reduction potentials of the compound II/ferric and compound I/compound II couples have been studied, using potassium hexachloroiridate as a mediator titrant, by thin-layer spectroelectrochemistry. Compound I, which is 2 equiv more oxidized than the ferric (i.e., resting) form of the enzyme, was reversibly formed via a compound II intermediate; no evidence for a ferric porphyrin pi-cation radical intermediate was obtained. At 25 degrees C, E degrees' (compound I/compound II) = 897.9 +/- 3 mV (NHE) and E degrees'-(compound II/ferric) = 869.1 +/- 2 mV. Redox thermodynamic parameters, obtained from the temperature dependences of the reduction potentials of both couples, are reported. The reaction entropies (delta S degrees rc) for the compound II/ferric and compound I/compound II couples are 19.8 +/- 3.9 and 12.1 +/- 3.7 eu, respectively. This result indicates that the reorganization energy for the macrocycle-centered couple is lower than that for the metal-centered one. Together with our observation that E degrees' for the former is ca. 30 mV greater than that for the latter, these results suggest that compound I is more reactive toward outer-sphere reductants than compound II. In particular, the electron self-exchange rates for the compound I/compound II and compound II/ferric couples are estimated to be 4.4 x 10(-1) and 4.9 x 10(-4) M-1 s-1, respectively. Surprisingly, the formation of compound I from ferric HRP is accompanied by an almost zero standard entropy (delta S degrees') change.

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