A biomimetic long-range electron transfer (ET) system consisting of the blue copper protein azurin, a tunneling barrier bridge, and a gold single-crystal electrode was designed on the basis of molecular wiring self-assembly principles. This system is sufficiently stable and sensitive in a quasi-biological environment, suitable for detailed observations of long-range protein interfacial ET at the nanoscale and single-molecule levels. Because azurin is located at clearly identifiable fixed sites in well controlled orientation, the ET configuration parallels biological ET. The ET is nonadiabatic, and the rate constants display tunneling features with distance-decay factors of 0.83 and 0.91 Å ؊1 in H2O and D2O, respectively. Redoxgated tunneling resonance is observed in situ at the single-molecule level by using electrochemical scanning tunneling microscopy, exhibiting an asymmetric dependence on the redox potential. Maximum resonance appears around the equilibrium redox potential of azurin with an on͞off current ratio of Ϸ9. Simulation analyses, based on a two-step interfacial ET model for the scanning tunneling microscopy redox process, were performed and provide quantitative information for rational understanding of the ET mechanism.blue copper protein ͉ scanning tunneling microscopy ͉ nanoscale bioelectronics ͉ bioelectrochemistry C harge transfer plays key roles in many chemical and biological processes as well as in molecular electronics (1-5). For example, long-range protein electron transfer (ET) is central in aerobic respiration and photosynthesis. The importance of longrange ET is illustrated by a recent special issue of PNAS on this topic (6-12). Several articles reflect broadly the current status of this subject. However, one of the major objectives in nanoscale science and technology is to fabricate molecular electronic devices with specified functions. Molecular electronics is rooted in the concept of molecular charge transfer (particularly, molecular conductivity) (13,14). Two essential steps involved in bottom-up manipulations are (i) organizing molecules into nanoscale structures and (ii) interfacing such nanostructures with macroscopically addressable components (e.g., metal and semiconductor electrodes). Molecular electronic device function thus rests fundamentally on charge transfer through organic and͞or biological molecules and across the interface between molecules and macroscopic electrodes (15, 16). Understanding of charge transfer mechanisms has mostly been based on average results of macroscopic measurements. The advent of scanning probe microscopies, along with other supersensitive techniques, has made it possible to characterize or directly observe charge transfer through organic molecules down to the nanoscale and single-molecule levels, as illustrated by measurements of singlemolecule conductivity (17-21) and probing of molecular switching and resonant tunneling (22,23). This, however, remains a daunting challenge for proteins, with difficulties arising from the assembly of suitable st...

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The recent expression of an azurin mutant where the blue type 1 copper site is replaced by the purple Cu A site of Paracoccus denitrificans cytochrome c oxidase has yielded an optimal system for examining the unique electron mediation properties of the binuclear Cu A center, because both type 1 and Cu A centers are placed in the same location in the protein while all other structural elements remain the same. Long-range electron transfer is induced between the disulfide radical anion, produced pulse radiolytically, and the oxidized binuclear Cu A center in the purple azurin mutant. The rate constant of this intramolecular process, k ET ‫؍‬ 650 ؎ 60 s ؊1 at 298 K and pH 5.1, is almost 3-fold faster than for the same process in the wild-type single blue copper azurin from Pseudomonas aeruginosa (250 ؎ 20 s ؊1 ), in spite of a smaller driving force (0.69 eV for purple Cu A azurin vs. 0.76 eV for blue copper azurin). The reorganization energy of the Cu A center is calculated to be 0.4 eV, which is only 50% of that found for the wild-type azurin. These results represent a direct comparison of electron transfer properties of the blue and purple Cu A sites in the same protein framework and provide support for the notion that the binuclear purple Cu A center is a more efficient electron transfer agent than the blue single copper center because reactivity of the former involves a lower reorganization energy.The Cu A centers (1) serve as the electron uptake site in the terminal respiratory enzyme cytochrome c oxidase (2) and also as a redox center in nitrous oxide reductase (N 2 OR) (3). A combination of x-ray structural characterization (4-7) and spectroscopic studies (for example, see refs. 8-17) on native enzymes, water-soluble fragments containing the Cu A center (18-20), engineered Cu A centers (21-23), and inorganic model compounds (24, 25) has established Cu A as a mixed valence [Cu(1.5) Ϫ Cu(1.5)] (S ϭ 1͞2) center with two copper ions in a Cu 2 S 2 diamond core, and these studies have provided a firm basis for understanding the structure and function of this class of biological copper centers. An immediate question that this unusual structure raised was what functional advantage has led to its selection, in particular compared with the type 1 (T1) blue copper centers. At least two distinct, though not mutually exclusive, rationales have been brought up so far. One is that the delocalized mixed-valence structure of the Cu A site would facilitate the unidirectional long-range electron transfer (ET) to the cytochrome a site of the enzyme (11,26). The other suggested that the Cu A structure would yield a lower reorganization energy, as the metal-ligand bond length changes upon ET would amount to only half of those occurring in a mononuclear site (11,14,27).To address the above question, ET studies on both the blue copper (28-31) and the purple Cu A proteins (26, 32-36) have been carried out. An ideal system to directly answer the above question will be a well-characterized protein where either the blue copper ...

The Cu(ll) sites of azurins, the blue single copper proteins, isolated from Pseudomonas aeruginosa and Akaligenes spp. (Iwasaki) are reduced by CO-radicals, produced by pulse radiolysis, in two distinct reaction steps: (i) a fast bimolecular phase, at the rates (5.0 ± 0.8) x l0o M-l s-' (P. aeruginosa) and (6.0 ± 1.0) x 104 M-l s-1 (Akaligenes); (ii) a slow unimolecular phase with specific rates of 44 ± 7 s'1 in the former and 8.5 ± 1.5 s-1 for the latter (all at 298 K, 0.1 M ionic strength). Concomitant with the fast reduction of Cu(II), the single disulfide bridge linking cysteine-3 to -26 in these proteins is reduced to the RSSR-radical ion as evidenced by its characteristic absorption band centered at 410 nm. This radical ion decays in a unimolecular process with a rate identical to that of the slow Cu(ll) reduction phase in the respective protein, thus clearly suggesting that a long-range intramolecular electron transfer occurs between the RSSRradicals and the Cu(ll) site. The temperature dependence ofthe internal electron transfer process in both proteins was measured over the 40C to 420C range. The activation parameters derived are AH* = 47.5 ± 4.0 and 16.7 ± 1.5 kJUmol'; and ASO = -56.5 ± 7.0 and -171 ± 18 J K-1 mol1, respectively.Using the Marcus theory, we found that the intramolecular electron transfer rates and their activation parameters observed for the two azurins correlate well with the distances between the reactive sites, their redox potential, and the nature of the separating medium. Thus, azurins with distinct structural and reactivity characteristics isolated from different bacteria or modified by site-directed mutagenesis can be used in comparing long-range electron transfer processes between their conserved disulfide bridge and the Cu(ll) sites.