A novel electrochemical system has been designed and assembled to study the kinetic activity of cytochrome c oxidase. Gold electrodes coated with 3-mercapto-1-propanol formed the surface for the physisorption of monolayers of cytochrome c and cytochrome c oxidase or a preformed cytochrome c−cytochrome c oxidase complex. The films were investigated by cyclic voltammetry at scanning at rates slow enough to permit near redox equilibrium between electrode and redox protein and hence obtain redox midpoint potentials. Cytochrome c monolayers alone displayed a reversible midpoint potential at pH 8 (E m8 vs NHE) at +240 mV, close to the native cytochrome c value observed in solution. In contrast, oxidase monolayers alone failed to support any detectable redox contact between electrode and protein, implying that the distances between the oxidase redox cofactors in the adsorbed oxidase are too far away from the electrode to promote significant electron transfer rates. However, adsorption of a preformed cytochrome c−cytochrome c oxidase complex promoted effective redox contact, demonstrating electron transfer with an apparent onset halfpoint potential at +225 mV. This effect is consistent with the mandatory requirement for cytochrome c to mediate electrons from the electrode to cytochrome c oxidase and presumably in a way reflecting the physiological pathway. Cyclic voltammetric measurements arranged to determine the rates of electron transfer between electrode and the complex showed that at scan rates up to 50 mV/s, extraordinary kinetic turnover is displayed attributable to the catalysis of oxygen reduction. Thus it is established that the protein complex can be assembled and enable the natural mediation of electron transfer from the electrode by cytochrome c to the enzyme at a rate fast enough for catalysis to be observed and controlled.

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.

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.

Free base protoporphyrin IX (PP), Zn(II)protoporphyrin IX (Zn(II)PP), Fe(III)protoporphyrin IX (Fe(III)PP), and Fe(III)protoporphyrin IX dimethylester (Fe(III)PPDME) as well as free base hematoporphyrin were selectively chemisorbed onto thiol-silanized quartz in aqueous media at neutral pH values and at room temperature. The self-assembled monolayers (SAMs) were characterized by UV/vis absorption and fluorescence spectroscopy. Evidence for chemisorption through the formation of thioether linkages between the immobilized thiols at the surface and the vinyl or the hydroxyethyl groups of the porphyrins is as follows:  (1) Only porphyrins that possess either vinyl or hydroxyethyl groups show the characteristic absorption and properties of a SAM; porphyrins free of vinyl or hydroxyethyl groups do not form SAMs. (2) Attachment to the surface through the propionates of the metal is ruled out since the presence of both metal and propionates is not mandatory for the formation of SAMs. (3) None of the porphyrins form SAMs on bare quartz or on quartz silanized with propyltrimethoxysilane. To our knowledge, this is the first description of a direct reaction under physiological conditions between thiols and the vinyl or the hydroxyethyl groups of porphyrin. This is analogous to the covalent attachment of heme in cytochrome c through the poorly understood reaction between the vinyl groups of the heme and the thiol of cysteine. Furthermore, this surface-promoted chemistry provides a simple and direct method for attaching unmodified porphyrins onto solid substrates to form remarkably robust monolayers. Linear dichroism revealed that the Fe(III)PPs in the monolayer are tilted in average at an angle of 33° relative to the substrate, shifting to 46° after ligation to imidazole. Carbon monoxide coordinates to Fe(II) in SAMs of FePP and FePPDME. SAMs of Fe(III)PP and Fe(III)PPDME ligate imidazole cooperatively and, after reduction, bind CO reversibly. In contrast, only one imidazole ligates to Zn(II)PP as a SAM, and no interaction with CO was observed.

To better understand the relation between structure and function of complex natural heme proteins, we have designed and synthesized de novo simplified versions called maquettes. Furthermore, we have assembled organized monolayers of these heme protein maquettes onto silanized quartz to more define their structural and functional properties and to take the first step in constructing robust devices that exploit biological chemistry. First, two di-α-helical peptides, α2, were designed and synthesized to self-assemble into a four-helix bundle [α2]2. The assembly has a well-developed hydrophobic interior and coordinates iron protoporphyrin IX (heme) by bis-histidine ligation. The chemisorption of these synthetic hemoproteins onto silanized quartz was achieved by reacting disulfide bridges in the loop region of each α2 with thiols immobilized at the surface. Self-assembled monolayers of synthetic hemoproteins were characterized by UV/vis spectroscopy. UV absorption of the tryptophan reveals the presence of peptides on the substrate, and circular dichroism (CD) seems to indicate that the axes of the α-helices are oriented at a angle less than 45° relative to the substrate. The construction of monolayers of hemoproteins was successfully achieved in two different ways: (1) Hemes were incorporated after the apoproteins were self-assembled onto the silanized quartz substrate. This process led to bis-histidyl ligated hemes and to physisorbed porphyrins inside or at the surface of the monolayer. Physisorbed hemes were removed by immersion of the film in NaCl and imidazole solutions. (2) Heme-containing holoproteins were prepared in solutions before self-assembly onto silanized quartz. Linear dichroism of the heme bis-ligated to the histidines showed an average tilt angle of the porphyrin plane of 40° relative to the surface, consistent with an inclination of the whole assembly on the substrate suggested by CD measurements. Monolayers of hemoproteins, after reduction, bind CO by displacing one of the histidines. Their absorption spectra are remarkably similar to the ones reported for the cytochrome c 3.

The rate constants of the quenching of the fluorescence of the title compound (DCA) have been measured in hexane solution with a variety of aromatic and aliphatic electron donors. The plots of the logarithm of the rate constants vs. the donors' ionization potentials show two distinct Rehm-Weller type functions, one for the ' 7 1 ' donors and another for the ' n ' donors. The difference in quenching efficiency of these two groups is tentatively explained according to the different electrostatic interaction terms, that for the ' n ' donors being more favourable.

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