We have surveyed proteins with known atomic structure whose function involves electron transfer; in these, electrons can travel up to 14 A between redox centres through the protein medium. Transfer over longer distances always involves a chain of cofactors. This redox centre proximity alone is sufficient to allow tunnelling of electrons at rates far faster than the substrate redox reactions it supports. Consequently, there has been no necessity for proteins to evolve optimized routes between redox centres. Instead, simple geometry enables rapid tunnelling to high-energy intermediate states. This greatly simplifies any analysis of redox protein mechanisms and challenges the need to postulate mechanisms of superexchange through redox centres or the maintenance of charge neutrality when investigating electron-transfer reactions. Such tunnelling also allows sequential electron transfer in catalytic sites to surmount radical transition states without involving the movement of hydride ions, as is generally assumed. The 14 A or less spacing of redox centres provides highly robust engineering for electron transfer, and may reflect selection against designs that have proved more vulnerable to mutations during the course of evolution.

This paper describes the use of surface plasmon resonance spectroscopy and self-assembled monolayers (SAMs) of alkanethiols on gold to evaluate the ability of surfaces terminating in different combinations of charged groups to resist the nonspecific adsorption of proteins from aqueous buffer. Mixed SAMs formed from a 1:1 combination of a thiol terminated in a trimethylammonium group and a thiol terminated in a sulfonate group adsorbed less than 1% of a monolayer of two proteins with different characteristics: fibrinogen and lysozyme. Single-component SAMs formed from thiols terminating in groups combining a positively charged moiety and a negatively charged moiety were also capable of resisting the adsorption of proteins. Single-component SAMs presenting single charges adsorbed nearly a full monolayer of protein.The amount of protein that adsorbed to mixed zwitterionic SAMs did not depend on the ionic strength or the pH of the buffer in which the protein was dissolved. The amount of protein that adsorbed to singlecomponent zwitterionic SAMs increased as the ionic strength of the buffer decreased; it also decreased as the pH of the buffer increased (at constant ionic strength). Single-component zwitterionic SAMs composed of thiols terminating in N,N-dimethyl-amino-propane-1-sulfonic acid (-N + (CH3)2CH2CH2CH2SO3 -) groups were substantially more effective at resisting adsorption of fibrinogen and lysozyme from buffer at physiological ionic strength and pH than single-component zwitterionic SAMs composed of thiols terminating in phosphoric acid 2-trimethylamino-ethyl ester (-OP(O)2 -OCH2CH2N + (CH3)3). Several of these zwitterionic SAMs were comparable to the best known systems for resisting nonspecific adsorption of protein.

The redox behavior of cytochrome c (cyt c) adsorbed to gold electrodes modified with self-assembled monolayers (SAMs) depends on the SAM. This paper examines SAMs generated from alkanethiols terminating in trimethylammonium (1), sulfonate (2), methyl (3), amine (4), and carboxylic acid (5) groups and from an aromatic thiol (6). The redox potentials of cyt c adsorbed on SAMs of 1 and 5 are relatively close to the formal potential of native cyt c measured in solution. The redox potentials of cyt c adsorbed on SAMs of 3, 4, and 6 are significantly shifted from the formal potential, and a reduction peak at about 0.5 V more negative than the formal potential (that is, a value corresponding to a more difficult reduction) was observed in all three cases. These observations suggest that cyt c changes its conformation significantly on adsorption on these surfaces. No redox peaks were observed for cyt c adsorbed on SAMs of 2, although surface plasmon resonance (SPR) studies indicate that the SAMs of 2 irreversibly adsorbed approximately a double layer of cyt c. Mixed SAMs were also studied. Most interestingly, cyt c adsorbed on mixed SAMs formed from the combinations of 1 and 2 exhibited significantly slower electron transfer (0.3-1.2 s -1 ) than cyt c adsorbed on a homogeneous SAM of 1 (45 s -1 ). These observations suggest changes in protein orientation due to the presence of the sulfonate groups at the interface. This study suggests that electrochemical measurement can be a useful probe for the conformation and orientation of protein adsorbed on surfaces.

An electrical junction formed by mechanical contact between two self-assembled monolayers (SAMs)sa SAM formed from an dialkyl disulfide with a covalently linked tetracyanoquinodimethane group that is supported by silver (or gold) and a SAM formed from an alkanethiolate SAM that is supported by mercurysrectifies current. The precursor to the SAM on silver (or gold) was bis(20-(2-((2,5-cyclohexadiene-1,4-diylidene)dimalonitrile))decyl)) disulfide and that for the SAM on mercury was HS(CH2)n-1CH3 (n ) 14, 16, 18). The electrical properties of the junctions were characterized by current-voltage measurements. The ratio of the conductivity of the junction in the forward bias (Hg cathodic) to that in the reverse bias (Hg anodic), at a potential of 1 V, was 9 ( 2 when the SAM on mercury was derived from HS(CH2)15CH3. The ratio of the conductivity in the forward bias to that in the reverse bias increased with decreasing chain length of the alkanethiol used to form the SAM on mercury. These results demonstrate that a single redox center asymmetrically placed in a metal-insulator-metal junction can cause the rectification of current and indicate that a fixed dipole in the insulating region of a metal-insulator-metal junction is not required for rectification.

The new artificial membrane provides an improved PAMPA model.

Iron(III) protoporphyrin IX (Fe(III)PP) and iron(III) hematoporphyrin (Fe(III)HP) were selectively and covalently attached to dimercaptoalkane-modified gold electrodes. Reaction of their vinyl or hydroxyethyl groups with the surface-immobilized thiols produced thioether linkages, reminiscent of the heme macrocycle attachment in c-type cytochromes. Cyclic voltammetry revealed reversible electrochemistry of self-assembled monolayers (SAMs) of FePPs and FeHPs on the thiol-modified gold substrates. The surface coverage estimated from the charges transferred corresponds to 30% of a monolayer. The heterogeneous rate constant of electron transfer between the Fe(III)PPs and the gold substrate decreases exponentially with the length of the spacer, demonstrating a value of 1.0 Å -1 for the tunneling length coefficient, β. At pH 8, a linear increase of the formal redox potential (E°′) with the length of the linker was also observed. This suggests that in the film, there is a close contact between the porphyrins and the alkane SAM: the E°′ is affected by the drop of the electrostatic potential from the electrode to the surface of the alkane SAM, and also that there is a strong ion pairing of the Fe(III)PPs in the film with the anions of the solution. The E°′ of Fe(III)PPs in the SAM shows a strong and complex dependency on the pH of the solution, explained by variations in the coordination of the iron, involving hydroxyl ions, water, and eventually dioxygen molecules. Interactions of the iron with either functional groups present at the surface of the substrate or with the propionate groups attached to the porphyrin ring, do not appear to be involved in the electron-proton transfer coupling mechanisms.

We present isotherm and X-ray reflectivity (XR) measurements from Langmuir monolayers of a de novo synthetic di-α-helical peptide, consisting of two identical 31-residue, mostly α-helical peptide units joined by a disulfide bond at their amino-termini. Fitting the XR data to slab models shows that the dihelices lie in the plane of the interface at low pressures. The monolayers were insufficiently stable for study at high pressures, but Langmuir films based on a derivative of the peptide alkylated at its amino termini permitted investigations over a larger range of pressures. We observed an orientational transition, in which the α-helices begin by lying in the plane of the interface at low surface pressures and orient themselves approximately normal to the interface at high pressures. We draw the same conclusions from the XR data when we analyze it using box refinement, an iterative, model-independent method for recovering structure from XR data. Mixtures of these palmitoylated peptides with a fatty acid (palmitic acid) or a phospholipid (DLPE) behaved similarly. None of the systems produced peaks in the grazing incidence diffraction signal indicative of long-range ordering of the upright α-helices. Off-specular in-plane scattering measurements based on the difference signal between the peptide/DLPE mixture and pure DLPE suggest that the peptide achieves only liquidlike order within the plane. We discuss the implications and prospects for future work on designed peptide monolayers incorporating prosthetic groups that could be used to study electron transfer in proteins and provide a basis for biomolecular electronics applications.

Experimental explorations of functional mechanisms in natural electron-transfer proteins are often frustrated by their fragility and extreme complexity. We have designed and synthesized four-R-helix-bundle redox proteins, maquettes, that are much simplified and more robust than natural redox proteins and can be designed to bind onto electrode surfaces to facilitate systematic investigations. The points of interest that can be now assessed are not only the processes that govern biological assembly of equilibrium structures, electrochemistry, and electron tunneling rates but also how these factors are coupled together to effect redox driven catalysis. Here we describe maquettes that bis-histidine ligate protoporphyrin IX (heme), much like native b cytochromes, as well as contain charged surface patches, much like native cytochrome c. The positively charged residues aid adsorption to negatively charged surfaces, such as gold electrodes modified by 11-mercaptoundecanoic acid, and facilitate cyclic voltammetry (CV) measurements. CV demonstrates the reversible electrochemistry typical for cytochrome b as well as the coupling of the b-heme oxidation and reduction to proton exchange. The pH dependency of redox midpoint potentials reveals a major (three pH units) shift of the pK a which matches the shift previously shown to originate in nearby glutamates 1 . The redox potentials correspondingly shift from -0.24 (pH > pK red , deprotonated) to -0.11 V (pH < pK ox , protonated). The rate of electron transfer at zero driving force between the hemes and the gold electrode was determined to be 120 s -1 , a rate consistent with tunneling through the mercaptoundecanoic acid spacer and suggesting that the coupled proton exchange is not rate limiting. Reduction of the heme in the presence of CO-saturated buffer shifted the oxidation peak from -0.2 to +0.35 V, indicating massive preferential CO binding to the reduced heme. Consistent with solution spectroscopy, CO must displace one axial histidine to the heme to form the His-CO form of the ferrous heme. The CO is released upon heme oxidation at high potentials. In contrast to coupled proton exchange, CO binding/release and ligand exchange are slow on the time scale of electron tunneling between the heme edge and the electrode.