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Robert J. P. Williams was a pioneer in advancing our understanding of the roles of chemical elements, especially the metals, in biology and in biological evolution. During the first half of his career of more than 60 years at Oxford University he studied the thermodynamic stabilities of transition-metal complexes with organic ligands, their redox properties, magnetism and colour, to understand their biological function. In parallel he collaborated with biologists and biophysicists, for example with Bert Vallee, studying zinc in proteins. Williams was the first to describe how proton gradients could be used to drive the formation of the universal biological fuel, ATP (adenosine triphosphate), a fundamental step in biological energetics. From the late 1960s he studied many proteins that use metal ions for catalysis, for electron transfer and cellular regulation. A leading figure in the establishment of the Oxford Enzyme Group, Williams developed high-field nuclear magnetic resonance (NMR) to study the mobility and dynamics of many protein structures, leading to a deeper understanding of protein function. He held the Royal Society Napier Research Professorship from 1974 until his retirement in 1991. Subsequently he published several books setting out his understanding of the roles of metal ions in biology, and their wider significance in evolution. Bob Williams's deep insights across many disciplines made him a charismatic teacher. His lateral style of thinking never failed to inspire. His legacy lies in the successful careers of his many students and collaborators worldwide and the vigour of the new discipline of bioinorganic chemistry that he helped to establish.

The direct (unmediated) electrochemistry of several redox proteins at pyrolytic graphite electrodes has been studied by square-wave voltammetry. For rubredoxin and the 2 left bracket 4Fe-4S right bracket ferrodoxin from Clostridium pasteurianum and flavodoxin from Megasphaera elsdenii rapid heterogeneous electron transfer is promoted by multivalent cations such as Mg**2** plus and Cr(NH//3)//6**3** plus . These proteins carry an excess of negatively charged residues, and it is suggested that their approach to the electrode surface is assisted by the high positive charge density in or near the outer Helmholtz plane that may be generated by multivalent cations. In addition, specific protein-cation association may be important. For azurin from Pseudomonas aerguginosa, direct electrochemistry is observable without the requirement of cation promoters.

The application of scanning probe methods to the high-resolution imaging of biological structure has been developing rapidly during the past few years. In contrast to diffraction and electron microscopy methods, imaging is direct and can be carried out under fluid. Scanning Tunneling Microscopy (STM) allows a resolution of electronic as well as topographic structure, and we are accordingly interested in its application to the high resolution imaging of metalloproteins. PARAGRAPH MARK REMOVED. Metallothionein has been imaged under buffered solution by in situ STM. The dumbbell morphology has been resolved and the metal centers appear to give rise to enhanced tunneling current.

The electrochemistry of a range of electron-transfer proteins at edge- and basal-plane graphite electrodes has been reconsidered using a microscopic model, which involves fast electron transfer at very small oxygen-containing electroactive surface sites. This model assumes that mass transport to the electrode occurs by radial diffusion when the density of the surface active sites is low (as is generally true in the case of the basal-plane graphite electrode) and by linear diffusion when the density of the active sites is increased sufficiently to cause overlap of the diffusion layers. With this model it is now proposed that the electrochemistry of cytochrome c, plastocyanin, and ferredoxin occurs with a very fast rate of charge transfer (≥1 cm s-1) at both edge- and basal-plane graphite electrodes. Critical factors, such as the mode of surface preparation (including covalent derivatization), the pH, and the presence in the electrolyte of cations such as Mg2+ or Cr(NH3)63+, control the density of surface sites, which result in the electrochemistry of a specific protein. This contrasts with the conclusion that has been reached previously based upon a conventional macroscopic model, which supposes that the rate of electron transfer is subject to enhancement or depression through these factors. The proposal that the electron-transfer process at the protein-graphite electrode interface is very fast over a wide range of conditions is now consistent with homogeneous kinetic studies where electron-transfer reactions of proteins, particularly amongst physiological partners, are also known to be fast. © 1989 American Chemical Society.

A surface cysteine-containing redox protein (azurin mutant S118C) has been labelled with a C60-based thiol-selective reagent leading to electrochemical interactions between the fullerene and protein redox centre.

The direct electrochemistry of redox proteins has been achieved at a variety of electrodes, including modified gold, pyrolytic graphite and metal oxides. Careful design of electrode surfaces and electrolyte conditions are required for the attainment of rapid and reversible protein-electrode interaction. The electron transfer reactions of more complex systems, such as redox enzymes, are now being examined. The 'well-behaved' electrochemistry of redox proteins can be usefully exploited by coupling the electrode reaction to enzymes for which the redox proteins act as cofactors. In systems where direct electron transfer is very slow, small electron carriers, or mediators, may be employed to enhance the rate of electron exchange with the electrode. The organometallic compound ferrocene and its derivatives have proved particularly effective in this role. A new generation of electrochemical biosensors employs ferrocene derivatives as mediators.

Our appreciation of the scholarly ideas and thinking of Bob Williams is illustrated here by a few of the areas in which he inspired us. His journey to bring inorganic chemistry to life began with an early interest in analytical chemistry, rationalising the relative stabilities of metal coordination complexes (The Irving-Williams Series), and elucidating the organometallic redox chemistry of vitamin B12. He (and Vallee) recognised that metal ions are in energised (entatic) states in proteins and enzymes, which themselves are dynamic structures of rods and springs. He played a key role in helping Rosenberg to pave the road toward the clinic for the anticancer drug cisplatin. He believed that evolution is not just dependent on DNA, but also on the metallome. Organisms and the environment are one system: does DNA code directly for all the essential elements of life?

Nitrite is converted to nitric oxide by haem or copper-containing enzymes in denitrifying bacteria during the process of denitrification. In designing an efficient biosensor, this enzymic turnover must be quantitatively assessed. The enzyme nitrite reductase from Alcaligenes faecalis contains a redox-active blue copper centre and a nonblue enzyme-active copper centre. It can be covalently tethered to modified gold-electrode surfaces in configurations in which direct electron transfer is possible. A surface cysteine mutant of the enzyme can be similarly immobilised on bare electroactive gold substrates. Under such circumstances, however, electron transfer cannot be effectively coupled with substrate catalytic turnover. In using either the natural redox partner, pseudoazurin, or ruthenium hexammine as an "electron-shuttle" or "conduit" between enzyme and a peptide-modified electrode surface, the coupling of electron transfer to catalysis can be utilised in the development of an amperometric nitrite sensor.

The influence of peptide-protein interactions on the electrochemistry of copper-containing pseudoazurin from Alcaligenes faecalis strain S-6 has been investigated by covalently binding cysteine-containing hexapeptides to a gold electrode surface. The hexapeptides contain three cysteines in the same positions with the remaining amino acids varied to give mixed charge (lysine, threonine, alanine), positive (lysine), overall neutral (alanine), and negative (glutamate) chemically modified electrode surfaces. These systematic variations in the amino acid sequence lead to large variations in voltammetric behavior for the Cu(II) --> Cu(I) heterogeneous pseudoazurin redox process encompassing fully reversible and diffusional, transitionally adsorbed, or strongly adsorbed forms of voltammetry. The variations in voltammetric behavior may be related to electrostatic interactions between the charges from the hexapeptide electrode modifiers and surface charges of pseudoazurin. A possible description of the pseudoazurin-electrode surface interaction is given.

Summary A scanning tunnelling microscopy study of adsorption of wild-type Pseudomonas putida putidaredoxin at a gold (111)–buffer interface has been made in real time. Reversible adsorption has been observed reflecting weak interaction of the wild-type protein with a gold (111) electrode. A genetically engineered mutant, C73S-D58C, which contains a surface thiol, has been used for ‘immobilization’ and ‘orientated adsorption’ on the gold surface. The implication of such orientated immobilization in development of a bio-electrode surface has been predicted.

Cytochrome P450BM3 is a self-sufficient fatty acid monooxygenase consisting of a diflavin (FAD/FMN) reductase domain and a heme domain fused together in a single polypeptide chain. The multidomain structure makes it an ideal model system for studying the mechanism of electron transfer and for understanding P450 systems in general. Here we report the redox properties of the cytochrome P450BM3 wild-type holoenzyme, and its isolated FAD reductase and P450 heme domains, when immobilized in a didodecyldimethylammonium bromide film cast on an edge-plane graphite electrode. The holoenzyme showed cyclic voltammetric peaks originating from both the flavin reductase domain and the FeIII/FeII redox couple contained in the heme domain, with formal potentials of −0.388 and −0.250 V with respect to a saturated calomel electrode, respectively. When measured in buffer solutions containing the holoenzyme or FAD-reductase domain, the reductase response could be maintained for several hours as a result of protein reorganization and refreshing at the didodecyldimethylammonium modified surface. When measured in buffer solution alone, the cyclic voltammetric peaks from the reductase domain rapidly diminished in favour of the heme response. Electron transfer from the electrode to the heme was measured directly and at a similarly fast rate (ks′ = 221 s−1) to natural biological rates. The redox potential of the FeIII/FeII couple increased when carbon monoxide was bound to the reduced heme, but when in the presence of substrate(s) no shift in potential was observed. The reduced heme rapidly catalysed the reduction of oxygen to hydrogen peroxide.

Soluble methane monooxygenase (sMMO) is a three-component enzyme that catalyses dioxygen- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Oxygenation occurs at the binuclear iron active centre in the hydroxylase component (MMOH), to which electrons are passed from NAD(P)H via the reductase component (MMOR), along a pathway that is facilitated and controlled by the third component, protein B (MMOB). We previously demonstrated that electrons could be passed to MMOH from a hexapeptide-modified gold electrode and thus cyclic voltammetry could be used to measure the redox potentials of the MMOH active site. Here we have shown that the reduction current is enhanced by the presence of catalase or if the reaction is performed in a flow-cell, probably because oxygen is reduced to hydrogen peroxide, by MMOH at the electrode surface and the hydrogen peroxide then inactivates the enzyme unless removed by catalase or a continuous flow of solution. Hydrogen peroxide production appears to be inhibited by MMOB, suggesting that MMOB is controlling the flow of electrons to MMOH as it does in the presence of MMOR and NAD(P)H. Most importantly, in the presence of MMOB and catalase, the electrode-associated MMOH oxygenates acetonitrile to cyanoaldehyde and methane to methanol. Thus the electochemically driven sMMO showed the same catalytic activity and regulation by MMOB as the natural NAD(P)H-driven reaction and may have the potential for development into an economic, NAD(P)H-independent oxygenation catalyst. The significance of the production of hydrogen peroxide, which is not usually observed with the NAD(P)H-driven system, is also discussed.

A new design for microelectrode array (MEA) devices fabricated by semiconductor-processing techniques is presented. The microelectrode surfaces consist of gold and are surrounded by an insulating silicon nitride layer. Each chip of these so-called Multi MEAs contains regular arrays with circular-shaped electrodes of eight different sizes: 1, 3, 5, 10, 50, 100, 500 and 1000μm. The Multi MEAs were electrochemically characterised by use of ferrocenecarboxylic acid. Well-defined cyclic voltammograms of the two small redox proteins, horse heart cytochrome c and anicyanin from Thiobacillus versutus, were obtained at variously surface-modified Multi MEAs. Furthermore, a very simple method to manufacture Multi MEAs with carbon surfaces is introduced.

The direct electrochemistry of the flavodehydrogenase domain of flavocytochrome b 2 engineered for l -mandelate dehydrogenase activity (FDH) has been investigated at an edge-plane pyrolytic graphite (EPG) electrode using poly- l -lysine as a promoter. Two redox couples (−0.481 and −0.605 V vs. SCE in Tris buffer solution at pH 7.5, scan rate 20 mV s −1 ) were obtained on the cyclic voltammogram which correspond to the separated two peaks in the one-electron reduction-reoxidation steps of enzyme bounded flavin mononucleotide (FMN). The electrochemical transformation of the substrate l -mandelic acid (LMA), catalysed by the FMN-domain of l -mandelate dehydrogenase (LMDH) is inhibited at bare or promoter-modified EPG, but both ferrocenemonocarboxylic acid (FMCA) and cytochrome c function as mediators.

Microelectrode array (MEA) devices were employed to investigate the direct electrochemistry of three redox proteins, namely: horse heart cytochrome c, amicyanin from Thiobacillus versutus and amicyanin from Paracoccus denitrificans. Cyclic voltammetric experiments were carried out with arrays of 15×15 square-shaped gold microdisks of 5 μm in dimension. The gold microelectrodes were defined by a photoresist or silicon nitride coating as the insulating layer. The occurrence of well-behaved, steady-state responses of the redox proteins only at MEAs fabricated with photoresist may be explained by a model which assumes that one component of the photoresist acts as a facilitator enabling electron transfer between the microelectrodes and the protein molecules. It could be shown that the photoresist facilitates the electrochemistry of positively and negatively charged proteins. The formal redox potentials of both types of amicyanin were studied over a range of solution pH. In agreement with previous studies, the formal redox potentials increased with decreasing pH because of the redox inactivity of amicyanin in the CuI state at low pH. These experiments demonstrated that photoresist-coated MEAs can be employed as versatile tools to gain insight into the properties of redox biomacromolecules.

CYP101 (cytochrome P450cam) catalyses the oxidation of camphor but has also been shown to catalyse the reductive dehalogenation of hexachloroethane and pentachloroethane. This reaction has potential applications in the biodegradation of these environmental contaminants. The hexachloroethane dehalogenation activity of CYP101 has been investigated by mutagenesis. The effects of active-site polarity and volume were probed by combinations of active-site mutations. Increasing the active-site hydrophobicity by the Y96A and Y96F mutations strengthened hexachloroethane binding but decreased the rate of reaction. Increasing the polarity with the F87Y mutation drastically weakened hexachloroethane binding but did not affect the rate of reaction. The Y96H mutation had little effect at pH 7.4, but weakened hexachloroethane binding while increasing the rate of dehalogenation by up to 40% at pH 6.5, suggesting that the imidazole side-chain was partially protonated at pH 6.5 but not at pH 7.4. Substitutions by bulkier side-chains at F87, T101 and V247 weakened hexachloroethane binding but increased the dehalogenation rate. The effect of the individual mutations was additive in multiple mutants, and the most active mutant for hexachloroethane reductive dehalogenation at pH 7.4 was F87W–V247L (80 min−1 or 2.5 × the activity of the wild-type). The results suggested that the CYP101 active site shows good match with hexachloroethane, the Y96 side-chain plays an important role in both hexachloroethane binding and dehalogenation, and hexachloroethane binding and dehalogenation places conflicting demands on active-site polarity and compromises were necessary to achieve reasonable values for both.

Carbon nanotubes, fullerene-related structures, have been used for the immobilisation of proteins and enzymes. We have been able to demonstrate, for the first time, direct imaging by high resolution transmission electron microscopy of Zn2Cd5-metallothionein, cytochromes c, c3, and β-lactamase I. This was achieved, without modification, because the biomolecules encapsulated within nanotubes appear to be shielded from the consequences of exposure to the intense electron beam. The results indicate that the internal surface of the nanotubes interacts strongly with the enzymes resulting in their immobilisation. In some cases, the proteins are seen to be distorted giving a concave meniscus inside the tubes. Single protein molecules, their dimers, tetramers and higher oligomers are observed inside the central cavity. Comparison of the catalytic activities of immobilised β-lactamase I on or in nanotubes with the free enzyme in the hydrolysis of penicillin, however, showed a significant amount of the immobilised enzyme remained catalytically active, implying that no drastic conformational change had taken place. The carbon nanotube appears to act as a benign host in its ability to encapsulate protein molecules within an environment which offers some protection. © 1998 Elsevier Science S.A. All rights reserved.

The in situ adsorption, under physiological conditions, of azurin molecules at a gold electrode surface has been monitored at the molecular level by scanning tunnelling microscopy. The introduction of free cysteine residues into the surface of the redox protein by structurally conservative mutagenesis allows the immobilisation to be controlled in a manner in which the protein electrochemical activity is retained.

A novel chiral redox-active ferrocene compound (FcVI) with amphiphilic properties has been synthesized. Cryogenic Transmission Electron Microscopy (cryo-TEM) has been used to estimate the shape and size of the FcVI aggregates in solution. Uni- and multi-lamellar vesicles (between 40 and 300 nm in diameter) were observed in water. Large particles (of more than 1 μm in diameter) with a hexagonal fine structure were found in 50 mM aqueous Na 2 SO 4 solution. Sonication transformed the latter into ‘rosette’-like structures. Cyclic voltammetry has been employed to investigate the electrochemical properties of FcVI. The amphiphile adsorbed on graphite electrodes and a reversible electrochemical behaviour, characteristic of ferrocene, was observed with redox potentials between 330 and 350 mV.

A novel chiral redox-active ferrocene compound (FcV1) with amphiphilic properties has been synthesised and used successfully to form protein + lipid ‘clusters’ with ferritin and gold-labelled BSA. These structures could be observed by cryogenic transmission electron microscopy and had diameters between 40 and 300 nm. It was shown that the novel lipid formed reasonably stable films on an edge plane graphite electrode surface and also mediated the electron transfer between flavocytochrome b 2 and this electrode. Consequently, the enzyme was incorporated into an FcV1 film and its response towards l (+)lactate in solution investigated. It could be shown that the electrode indeed was sensitive to l (+)lactate.

A ruthenocene-based surfactant (RcC12) has been synthesized. Its critical micelle concentration (CMC) and electrochemical behaviour have been investigated and cryogenic transmission electron microscopy has been used to estimate the shape and size of the novel metallo-micelles. The CMC of RcC12 was 0.4 mM in aqueous 0.1 M NaCl solution and micelles of a diameter between 5 and 10 nm were observed under the microscope. RcC12 underwent irreversible oxidation leading to the formation of a new electroactive species. RcC12 micelles incorporated water-insoluble compounds and made electrochemical investigations of those compounds possible in aqueous solution.

The electrochemistry of cytochrome b562 from E. coli has been studied at graphite and gold electrodes. At edge plane graphite surfaces neomycin promotes reversible, linear-diffusion limited electrochemistry across the pH range 4.5 to 8.0. At gold surfaces, a similar, but much more stable response is observed when the electrode is electrochemically modified with the cationic hexapeptide, KCTCCA. This reversible direct electrochemical response has been used to study the reduction potential of wild type cytochrome b562 as a function of pH, ionic strength and temperature. The enthalpic and entropic parameters for the redox equilbbrium show a dependence on pH consistent with the idea that the protonation of the haem propionate groups contribute significantly to the control of reduction equilibrium. The electrochemical response from the wild type protein is not reversible above pH 8 and is not detectable above pH 9. This is rationalised by the presence of a species with a deprotonated histidine ligand in the ferricytochrome but surprisingly, we have not observed any electrochemistry attributable to this species under the conditions employed in this work. We have also studied variants of this cytochrome which have bis-methionine ligation and undergo redox linked ligation state changes between low-spin and high-spin ferric species. The consequences of these changes for the electrochemical response are examined.

The development of the interest in bioelectrochemistry, from that of redox proteins and enzymes to the use in analyses, is described. Some of the difficulties that arose during this exercise are described.

In this study sonovoltammetry, voltammetry in the presence of power ultrasound, is used to investigate aspects of cytochrome c redox processes at conventional-sized electrodes under extreme mass transport conditions comparable to those found in microelectrode steady state experiments. A new small volume sonovoltammetry cell (10-20 mL) equipped with a high-intensity 3 mm diameter titanium horn was built and characterized. By variation of the electrode-to-horn separation, a range of accessible average diffusion layer thicknesses from typically 1 to 7 μm was determined and an approximate mass transport model based on a "uniformly accessible electrode" is suggested. On gold electrodes modified with 4,4′-bipyridyl disulfide, well-defined "steady state" sonovoltammograms for the reduction of ferricytochrome c corresponding to very fast electron transfer (ks > 0.1 cm s-1) were obtained, although the activity of the electrode surface was found to be sensitive to the applied potential and to some degree to the period and intensity of insonation. A model based on the adsorption of cytochrome c on bare gold and surface modified gold [Szucs et al. Electrochim. Acta 1992, 37, 403] is used to explain irreversible and reversible electrode deactivation processes. Voltammograms obtained on glassy carbon, basal, and edge plane pyrolytic graphite in the presence of ultrasound under high mass transport conditions were ill-defined possibly due to competing adsorption of impurities or the effect of high shear forces induced by ultrasound. © 1996 American Chemical Society.

We have generated mutants of cytochrome b562 in which the histidine ligand to the heme iron (His102) has been replaced by a methionine. The resulting proteins can have bis-methionine coordination to the heme iron, but the stability of this arrangement is dependent on oxidation state and solution pH. We have used optical, MCD, and EPR spectroscopies to study the nature of the heme coordination environment under a variety of conditions. Optical spectra of the reduced state of the single variant, H102M, are consistent with bis-methionine ligation. In its oxidized state, this protein is high-spin under all conditions studied, and the spectroscopic properties are consistent with only one of the methionine ligands being coordinated. We cannot identify what, if anything, provides the other axial ligand. A double variant, R98C/H102M (in which the heme is covalently attached to the protein through a c-type thioether linkage), is also bis-methionine coordinated in the ferrous state, but has significantly different properties in the oxidized state. With a pKa of 7.1 at 20 degrees C, the protein converts from a low-spin, 6-coordinate heme protein at low pH, to a high-spin species, similar to the high-spin species observed for the single variant. Our spectroscopic data prove that the low-spin species is bis-methionine coordinated. The reduction potential of this bis-methionine species has been measured using direct electrochemical techniques and is +440 mV at pH 4.8. The electrochemistry of these proteins is complicated by coupled coordination-state changes. Proof that the ferrous state is bis-methionine coordinated is provided by NMR results presented in the following paper.

The enlightened formation, by Bert and Kuggie Vallee, of a procedure whereby senior scientists spend short time of about a month at Harvard, Oxford and other institutions, is illustrated by the views and opinions of those selected.

The heme iron in cytochrome c-550 from Thiobacillus versutus has a methionine and a histidine as axial ligands. In order to study the characteristics of a possible lysine-histidine ligation in a heme protein, the methionine has been replaced by a lysine. This residue acts as a ligand between pH 3 and 12. The midpoint potential of the mutant has shifted -329 mV compared to wild type, but apart from this shift the pH dependence of the midpoint potential is unchanged, suggesting that the large drop is caused by specific ligand effects and not by protein refolding. While the EPR spectrum of wild-type cytochrome c-550 shows one species with gz = 3.35, in the spectrum of the mutant two species occur with gz values of 3.53 and 3.30. The intensity ratio of both species depends on the presence of organic cosolvents. In the low frequency region (-4 to -1 ppm) of the 1H NMR spectrum of mutant ferrocytochrome c-550, four one-proton peaks replace the resonances of the ligand methionine side chain protons. Using two-dimensional NMR spectroscopy (COSY and NOESY), these protons and five others have been assigned to the lysine ligand. The spectroscopic results obtained for this mutant show similarities with those observed for the alkaline form of cytochrome c, supporting the Lys-His ligation proposed for this protein. The data are consistent with the evidence for amine ligation in cytochrome f: the EPR spectrum of M100K cytc-550 is similar to that of cytochrome f. However, the NMR spectra show significant differences.(ABSTRACT TRUNCATED AT 250 WORDS)

To elucidate the kinetic properties of the methylamine dehydrogenase (MADH) redox chain of Thiobacillus versutus the reduction of cytochrome c-550 by MADH and amicyanin has been studied. Under steady state conditions, the rate constants of the reactions have been determined as a function of the ionic strength, both for wild type cytochrome c-550 and for mutants in which the conserved residue Lys14 has been replaced as follows: Lys14-->Gln (mutant [K14Q]cytochrome c-550) and Lys14-->Glu (mutant [K14E]cytochrome c-550). The second-order rate constant of the reduction of cytochrome c-550 by MADH shows a biphasic ionic-strength dependence. At low ionic strength the rate constant remains unchanged (wild type) or increases ([K14Q]cytochrome c-550) with increasing ionic strength, while at high salt concentrations the rate constant decreases monotonically as the ionic strength increases. It is suggested that conformational freedom exists in the association complex and that this is favourable for electron transfer. [K14Q]cytochrome c-550 and [K14E]cytochrome c-550 are reduced at rates 20-fold and 500-fold slower than wild-type cytochrome c-550 by MADH, due to a lower association constant. It is concluded that MADH possesses a negative patch with which cytochrome c-550 associates. Lys14 plays an important role in the formation of the reaction complex. The midpoint potentials of wild-type and mutant cytochrome c-550 have been determined by using cyclic voltammetry. [K14Q]cytochrome c-550 and [K14E]cytochrome c-550 show an increase in E0 of only 2 mV and 8 mV, respectively, compared to wild-type cytochrome c-550 (241 mV at pH 8.1). [K14Q]cytochrome c-550 and [K14E]cytochrome c-550 cytochrome c-550 are reduced by amicyanin at rates that are only slightly faster than for wild-type cytochrome c-550. The difference is partly attributable to the change in E0. High ionic strength results in a threefold increase in the rate in all three cases. These results indicate that charge interactions do not play a major role in the formation of the amicyanin/cytochrome c-550 reaction complex, suggesting an interaction at the hydrophobic patch of amicyanin. The reduction of cytochrome c-550 by MADH can be inhibited by Zn(2+)-substituted amicyanin. Ag(+)-amicyanin, however, has little effect on the reduction rate. These results suggest that MADH has a much higher affinity for Cu(2+)-amicyanin (substrate) than for Cu(+)-amicyanin (product). On the basis of these findings the roles of the components of the MADH redox chain are discussed.

A steady quasi-reversible electrochemical response due to parsley plastocyanin is reported at an edge-plane graphite electrode without the requirement for so-called adjunct-promoters. This observation is unusual in that studies involving plastocyanins from other sources require the addition of polyvalent cations to the solution in order to promote the heterogeneous electron transfer. Here cationic adjunct-promoters are required only under conditions of low ionic strength, where sigmoidal responses are obtained, characteristic of the presence of a low number of electroactive sites on the electrode surface such that conditions of radial, as opposed to linear, diffusion apply.

The development of the interest in bioelectrochemistry, from that of redox proteins and enzymes to the use in analyses, is described. Some of the difficulties that arose during this exercise are described.

The mass transport mechanism associated with the reduction of cytochrome c at gold electrodes modified by adsorption of 4,4'-bipyridyl disulfide (SS-bpy) has been investigated at a range of electrode configurations. Radial diffusion to the small electroactive sites on the electrode surface leads to the observation of sigmoidal shaped curves when the surface density of the modifier is low at a stationary conventionally sized gold electrode. High modifier coverage causes overlap of the diffusion layers which leaves linear diffusion as the dominant mode of mass transport resulting in peak-shaped cyclic voltammograms

A simple, fast and effective method for the immobilization of glucose oxidase onto a modified basal plane pyrolytic graphite electrode is described. To enhance the rate of electron exchange with the electrode, a ferrocene derivative was used as a mediator. The stability of the enzyme electrode was evaluated using direct current cyclic voltammetry.

A new type of microelectrode array device has been developed using semiconductor-processing techniques. An array of 15 × 15 square electrodes as small as 1 μm, spaced 100 μm apart, has been fabricated on a silicon chip of dimensions 1.5 × 1.5 mm2. Steady state electrochemistry was performed using these devices in both aqueous and non-aqueous media.

Direct electrochemistry of site-specific mutants of yeast iso-1-cytochrome c (cyt c) and their complexes with bovine cytochrome b5 (cyt b5) has been investigated at edge-plane pyrolytic graphite (EPG) and bis(4-pyridyl)-disulphide-modified gold electrodes. Structure/function relationships have been investigated with the particular aim of clarifying the factors controlling the interactions of proteins at electrode/electrolyte interfaces and the determinants for direct electrochemistry in ternary protein/protein/electrode adducts, e.g. cyt c/cyt b5/EPG. Investigations of the cyt c mutants alone revealed a variety of electrochemical responses: all the mutants show similar voltammetric reversibility at modified gold electrodes, whereas at EPG electrodes the reversibility follows the order: Asn52Ile-Cys102Thr greater than Cys102Thr greater than Asn52Ala-Cys102Thr. Mid-point potentials follow the order: Arg13Ile (+60 +/- 5 mV vs. standard calomel electrode) greater than Cys102Thr (+40 +/- 5 mV) greater than Lys27Gln (+30 +/- 5 mV) approximately Lys72Asp (+30 +/- 5 mV) greater than Asn52Ala-Cys102Thr (+15 +/- 5 mV) greater than Asn52Ile-Cys102Thr (-10 +/- 5 mV). The structural basis for these differences is briefly discussed. When these mutants are bound to cyt b5, the differences in electrochemical response are greatly enhanced in the ternary cyt c/cyt b5/EPG adducts. A minimal analysis of these differences supports a model of multiple overlapping binding and recognition domains on cyt c which may be finely tuned to allow ternary complex formation so that a single-site variation could modify or abolish direct electrochemistry in the ternary adduct.

Etude de l'adsorption d'une plastocyanine reduite sur une electrode d'or modifiee par un complexe isothiocyanato de chrome (III)

Herein we report two examples of the direct electrochemistry of cytochrome c and cytochrome b 5 to BPG electrodes, modified with lauric acid and laurylamine, respectively. Presumably the hydrophobic chain of modifier is bound to the graphite electrode, allowing the hydrophilic groups to interact with the charged residues carried by cytochrome c or cytochrome b 5

In anaerobic environments the first electron transfer in substrate-free P450cam is known to be thermodynamically unfavourable, but in the presence of dioxygen the reduction potential for the reaction shifts positively to make electron transfer thermodynamically favourable. Nevertheless a slower rate of electron transfer is observed in the substrate-free P450cam compared to substrate-bound P450cam. The ferric haem centre in substrate-free P450cam changes from six co-ordinate to five co-ordinate when reduced whereas in substrate-bound P450cam the iron centre remains five co-ordinate in both oxidation states. The slower rate of electron transfer in the substrate-free P450cam is therefore attributed to a larger reorganisation energy as predicted by Marcus theory.

The electron transfer reactions of four small redox proteins, cytochrome c. ferredoxin, plastocyanin and azurin, have been investigated at novel peptide-modified gold electrodes. These proved to be effective and selective in facilitating electron transfer. Good, quasi-reversible electron transfer was achieved selectively at different peptide-protein configurations by changing the pH or the ionic strength of the solution. The use of peptides as promoters for protein electrochemistry opens up the possibility of designing very specific electrode surfaces for larger molecules like enzymes.

The direct electrochemistry of the cytochrome c/cytochrome b5 and cytochrome c/plastocyanin complexes has been investigated at edge-plane graphite and modified gold electrode surfaces, which are selective for one of the two components of the complex. Electrochemical response of one protein at an otherwise electrostatically unfavorable electrode surface was achieved in the presence of the other protein, and the calculated heterogeneous electron-transfer rate constant and diffusion coefficient were found to be in good agreement with the values determined previously from the electrochemistry of the individual proteins [Armstrong, F. A., Hill, H. A. O., & Walton, N. J. (1988) Acc. Chem. Res. 21, 407 and references therein]. A dynamic model of the protein-protein-electrode ternary complex is proposed to explain the promotion effect, and this model is supported by a study comparing the electrochemical responses of covalent and electrostatic cytochrome c/plastocyanin complexes. It is also suggested that the behavior of protein-protein complexes at electrode surfaces could be related to that of the complexes associated with biological membranes.

The electrochemistry of the enzyme, sulfide:cytochrome c oxidoreductase, also known as flavocytochrome c552 from the purple sulfur bacterium, Chromatium vinosum, has been studied using several modified electrodes. Direct electron transfer between the heme of the flavocytochrome and an electrode is observed in the presence of a redox-inactive cationic species which promotes the voltammetry of the enzyme. Quasi-reversible electron transfer was achieved using the aminoglycoside, neomycin, as a promoter at either a modified gold or polished edge-plane graphite electrode. Further evidence for direct electron transfer is provided by the catalytic response of the enzyme at the electrode in the presence of substrate. Also reported is the direct spectroelectrochemistry of flavocytochrome c552 at an optically transparent thin layer gold electrode modified with Cys-Glu-Cys in the presence of neomycin.