A central vision in molecular electronics is the creation of devices with functional molecular components that may provide unique properties. Proteins are attractive candidates for this purpose, as they have specific physical (optical, electrical) and chemical (selective binding, self-assembly) functions and offer a myriad of possibilities for (bio-)chemical modification. This Progress Report focuses on proteins as potential building components for future bioelectronic devices as they are quite efficient electronic conductors, compared with saturated organic molecules. The report addresses several questions: how general is this behavior; how does protein conduction compare with that of saturated and conjugated molecules; and what mechanisms enable efficient conduction across these large molecules? To answer these questions results of nanometer-scale and macroscopic electronic transport measurements across a range of organic molecules and proteins are compiled and analyzed, from single/few molecules to large molecular ensembles, and the influence of measurement methods on the results is considered. Generalizing, it is found that proteins conduct better than saturated molecules, and somewhat poorer than conjugated molecules. Significantly, the presence of cofactors (redox-active or conjugated) in the protein enhances their conduction, but without an obvious advantage for natural electron transfer proteins. Most likely, the conduction mechanisms are hopping (at higher temperatures) and tunneling (below ca. 150-200 K).

The dynamics of protein molecules in the subnanosecond and nanosecond time range were investigated by time-resolved fluorescence polarization spectroscopy. Synchrotron radiation from a storage ring was used as a puked light source to excite the single tryptophan residue in a series of proteins. The full width at half maximum of the detected light pulse was 0.65 nsec, making it feasible to measure emission anisotropy kinetics in the subnanosecond time range and thereby to resolve internal rotational motions. We have carried out time-resolved emission anisotropy studies of the tryptophan fluorescence of a series of proteins to determine the angular range and kinetics of internal rotational motions of this chromophore. Nanosecond emission anisotropy studies have previously provided information concerning the segmental flexibility of domains of immunoglobulins (7, 8) and myosin (9). These studies had a time resolution of several nanoseconds and used extrinsic fluorescent probes. By using synchrotron radiation, we are now able to monitor directly the rotational motions of tryptophan residues in proteins, obviating any perturbations that may be caused by the insertion of an extrinsic probe. The distinctive properties of synchrotron radiation for these studies are its subnanosecond pulse width, high repetition rate and reproducibility, and high intensity in the ultraviolet region (10, 11). Proteins with a single tryptophan residue were studied because the interpretation of their emission anisotropy kinetics is more definitive than for proteins with multiple tryptophans. The molecules investigated were human serum albumin (69,000 daltons) (12), Staphylococcus aureus nuclease B (20,000 daltons) (13), human basic Al myelin protein (18,000 daltons) (14), and Pseudomonas aeruginosa holoazurin and apoazurin (14,000 daltons) (15). THEORY AND ANALYSISIn a time-resolved emission anisotropy experiment, an isotropic sample is excited by a pulse of y-polarized (vertically polarized) light, which produces an ensemble of preferentially aligned excited molecules. The orientations of the excited molecules then become randomized by rotational Brownian motion. For a fluorescent chromophore in a macromolecule, the rate of randomization depends both on the degree of flexibility of this group with respect to the macromolecule and on the size, shape, and internal motions of the macromolecule. These rotational motions can be monitored by measuring y(t) and x(t), the intensities of the y-polarized and x-polarized (horizontally polarized) components of the fluorescence emission as a function of time (for reviews, see refs. 16 and 17). The total fluorescence intensity F(t) and the emission anisotropy A(t) are defined by F(t) = y(t) + 2x(t)The simplest case is a chromophore with a single excited-state lifetime rotating in common with a rigid sphere. F(t) and A(t) are then given by[41 in which Fo is the initial fluorescence intensity and r is the excited state lifetime. For a rigid sphere, the rotational correlation time . is given by...

Electronic coupling to electrodes, Γ, as well as that across the examined molecules, H, is critical for solid-state electron transport (ETp) across proteins. Assessing the importance of each of these couplings helps to understand the mechanism of electron flow across molecules. We provide here experimental evidence for the importance of both couplings for solid-state ETp across the electron-mediating protein cytochrome c (CytC), measured in a monolayer configuration. Currents via CytC are temperature-independent between 30 and ∼130 K, consistent with tunneling by superexchange, and thermally activated at higher temperatures, ascribed to steady-state hopping. Covalent protein-electrode binding significantly increases Γ, as currents across CytC mutants, bound covalently to the electrode via a cysteine thiolate, are higher than those through electrostatically adsorbed CytC. Covalent binding also reduces the thermal activation energy, E a , of the ETp by more than a factor of two. The importance of H was examined by using a series of seven CytC mutants with cysteine residues at different surface positions, yielding distinct electrode-protein(-heme) orientations and separation distances. We find that, in general, mutants with electrode-proximal heme have lower E a values (from high-temperature data) and higher conductance at low temperatures (in the temperatureindependent regime) than those with a distal heme. We conclude that ETp across these mutants depends on the distance between the heme group and the top or bottom electrode, rather than on the total separation distance between electrodes (protein width).bioelectronics | temperature dependence | protein conduction

Examination of the optical and EPR properties of the ferric form of the iron containing superoxide dismutase from E. coli B, at pH values ranging from 4.5 to 10.9, has revealed two reversible structural transitions affecting the Fe:" ion. The apparent pK. values of these transitions are 5.1 ±0.3 and 9.0±0.3. The binding of azide has been studied over the pH range 4.5 to 10.7; the affinity of the Fe" for N~is independent of pH from 4.5 to -7.5, after which the dissociation constant decreased hy a factor of 10 per unit increase in pH. The apparent pK. which affects N~binding to the iron is 8.6±0.2. The association of N.~with the iron has been examined using the temperature-jump method at pH 7.4 and 9.3. The kinetics of ligand association were shown to conform to the minimal mechanism:

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