Electron transfer and electrocatalytic properties of the immobilized methionine80alanine cytochrome c variant

The M80A variant of yeast iso-1-cytochrome c (cytc), which features a noncoordinating Ala residue in place of the axial heme iron Met ligand, was chemisorbed on a gold electrode coated with 4-mercaptopyridine or carboxyalkanethiol self-assembled monolayers (SAM) and investigated by cyclic voltammetry at varying conditions of temperature, pH, and O2 concentration. The E degrees ' value (standard reduction potential for the heme Fe(III)/Fe(II) couple) of M80A cytc on both SAMs is of approximately -200 mV (vs the standard hydrogen electrode, SHE) at pH 7, which is more than 400 mV lower than that of native cytochrome c in the same conditions. The thermodynamics of Fe(III) to Fe(II) reduction and the kinetics of heterogeneous electron transfer (ET) are dominated by the presence of a hydroxide ion as the sixth axial heme iron ligand above pH 6. On both SAMs, protonation of the bound hydroxide ion is mainly responsible for the changes in these parameters at low pH, since the distances of ET between the heme and the electrode are found to be independent of pH in the range of 5-11. The invariance of the electrochemical features up to pH 11 indicates that no changes in heme iron coordination occur at high pH, at variance with native cytc. Most notably, immobilized M80A cytc is found to act as an efficient biocatalyst for O2 reduction from pH 5 to 11.0. This finding makes M80A cytc a suitable candidate as a constituent of a biocatalytic interface for O2 biosensing and opens the way for the exploitation of engineered cytochrome c in the bio-based detection of chemicals of environmental and clinical interest.     

Control of cytochrome C redox potential: axial ligation and protein environment effects

Axial iron ligation and protein encapsulation of the heme cofactor have been investigated as effectors of the reduction potential (E degrees ') of cytochrome c through direct electrochemistry experiments. Our approach was that of partitioning the E degrees ' changes resulting from binding of imidazole, 2-methyl-imidazole, ammonia, and azide to both cytochrome c and microperoxidase-11 (MP11), into the enthalpic and entropic contributions. N-Acetylmethionine binding to MP11 was also investigated. These ligands replace Met80 and a water molecule axially coordinated to the heme iron in cytochrome c and MP11, respectively. This factorization was achieved through variable temperature E degrees ' measurements. In this way, we have found that (i) the decrease in E degrees ' of cytochrome c due to Met80 substitution by a nitrogen-donor ligand is almost totally enthalpic in origin, as a result of the stronger electron donor properties of the exogenous ligand which selectively stabilize the ferric state; (ii) on the contrary, the binding of the same ligands and N-acetylmethionine to MP11 results in an enthalpic stabilization of the reduced state, whereas the entropic effect invariably decreases E degrees ' (the former effect prevails for the methionine ligand and the latter for the nitrogenous ligands). A comparison of the reduction thermodynamics of cytochrome c and the MP11 adducts offers insight on the effect of changing axial heme ligation and heme insertion into the folded polypeptide chain. Principally, we have found that the overall E degrees ' increase of approximately 400 mV, comparing MP11 and native cytochrome c, consists of two opposite enthalpic and entropic terms of approximately +680 and -280 mV, respectively. The enthalpic term includes contributions from both axial methionine binding (+300 mV) and protein encapsulation of the heme (+380 mV), whereas the entropic term is almost entirely manifest at the stage of axial ligand binding. Both terms are dominated by the effects of water exclusion from the heme environment.     

Direct electrochemistry of a bacterial sulfite dehydrogenase

Sulfite dehydrogenase from Starkeya novella is an alphabeta heterodimer comprising a 40.6 kDa subunit (containing the Mo cofactor) and a smaller 8.8 kDa heme c subunit. The enzyme catalyses the oxidation of sulfite to sulfate with the natural electron acceptor being cytochrome c550. Its catalytic mechanism is thought to resemble that found in eukaryotic sulfite oxidases. Using protein film voltammetry and redox potentiometry, we have identified both Mo- and heme-centered redox responses from the enzyme immobilized on a pyrolytic graphite working electrode: E m,8 (Fe III/II) +177 mV; E m,8 (Mo VI/V) +211 mV and E m,8 (Mo V/IV) -118 mV vs NHE; Upon addition of sulfite to the electrochemical cell a steady-state voltammogram is observed and an apparent Michaelis constant (Km) of 26(1) microM was determined for the enzyme immobilized on the working electrode surface, which is comparable with the value obtained from solution assays.     

Direct electrochemistry and bioelectrocatalysis of hemoglobin immobilized on carbon black

It is reported for the first time that hemoglobin (Hb) was immobilized on the surface of carbon black powders modified at the surface of a glassy carbon electrode. The cyclic voltammetric results showed that the immobilized Hb could undergo a direct quasi-reversible electrochemical reaction. Its formal potential, E(0), is -0.330 V in phosphate buffer solution (pH 6.9) at a scan rate of 100 mV/s and is almost independent of the scan rate in the range of 40-200 mV/s. The dependence of E(0), on the pH of the buffer solution indicated that the conversion of Hb-Fe(III)/Hb-Fe(II) is a one-electron-transfer reaction process coupled with one-proton-transfer. The experimental results also demonstrated that the immobilized Hb retained its bioelectrocatalytic activity for the reduction of H(2)O(2). Furthermore, the immobilized Hb can be stored at 4 degrees C for several weeks without any loss of the enzyme activity. Thus, the immobilized Hb may be used as a biocathodic catalyst in biofuel cells.     

pH-Induced changes in adsorbed cytochrome c. voltammetric and surface-enhanced resonance Raman characterization performed simultaneously at chemically modified silver electrodes

The influence of pH on the redox properties of cytochrome c (cyt c) adsorbed on roughened silver electrodes chemically modified with a self-assembled monolayer (SAM) of 11-mercapto-1-undecanoic acid (MUA) was studied with voltammetric techniques in combination with surface-enhanced resonance Raman scattering (SERRS). The experiments were performed simultaneously on the same electrode sample in a homemade spectroelectrochemical cell suitable for such applications. At pH 7.0 cyt c was found in its native state; at higher pH values (ranging from 8.0 to 9.0) the redox properties of the adsorbed protein varied considerably, featuring a redox behavior which does not resemble the one reported for the alkaline transition. Our results instead indicate the presence of an electrochemically inactive 6cLS species immobilized on MUA at pH 9.0. The pH-induced conformational changes observed for cyt c immobilized on the SAM of MUA were found to be repeatable and chemically reversible, meaning that the recovery of the electrochemical signal due to the native protein occurred instantaneously (on the second time scale) when the electrode was switched back to pH 7.0. The pH-induced changes observed were attributed to a conformational change involving a heme reorientation with respect to the electrode surface.     

Structural model for an alkaline form of ferricytochrome C

An (15)N-enriched sample of the yeast iso-1-ferricytochrome c triple variant (Lys72Ala/Lys79Ala/Cys102Thr) in an alkaline conformation was examined by NMR spectroscopy. The mutations were planned to produce a cytochrome c with a single conformer. Despite suboptimal conditions for the collection of spectra (i.e., pH approximately equal to 11), NMR remains a suitable investigation technique capable of taking advantage of paramagnetism. 76% of amino acids and 49% of protons were assigned successfully. The assignment was in part achieved through standard methods, in part through the identification of groups maintaining the same conformation as in the native protein at pH 7 and, for a few other residues, through a tentative analysis of internuclear distance predictions. Lys73 was assigned as the axial ligand together with His18. In this manner, 838 meaningful NOEs for 108 amino acids, 50 backbone angle constraints, and 203 pseudocontact shifts permitted the convergence of randomly generated structures to a family of conformers with a backbone RMSD of 1.5 +/- 0.2 A. Most of the native cytochrome c conformation is maintained at high pH. The NOE pattern that involves His18 clearly indicates that the proximal side of the protein, including the 20s and 40s loops, remains essentially intact. Structural differences are concentrated in the 70-80 loop, because of the replacement of Met80 by Lys73 as an axial ligand, and in the 50s helix facing that loop; as a consequence, there is increased exposure of the heme group to solvent. Based on several spectral features, we conclude that the folded polypeptide is highly fluxional.     

Direct electrochemistry and electrocatalysis of myoglobin immobilized on L-cysteine self-assembled gold electrode

Myoglobin (Mb) has been successfully immobilized on a self-assembled monolayer (SAM) of L-cysteine (Cys) on a gold electrode, Au/Cys. The presence of a pair of well-defined and nearly reversible waves centered at ca. 0.086 V vs Ag/AgCl (pH 6.5) suggests that the native character of Mb heme Fe(III/II) redox couple has been obtained. The formal potential of Mb on Cys SAM exhibited pH-dependent variation in the pH range of 5-9 with a slope of 55 mV/pH, indicating that the electron transfer is accompanied by a single proton exchange. Thermodynamic and kinetic aspects of Mb adsorption processes on Au/Cys were studied by using voltammetric and quartz-crystal microbalance methods. The Au/Cys electrode with immobilized Mb exhibited electrocatalytic activity toward ascorbic acid (AA) oxidation with an overpotential decrease of over 400 mV and a linear dependence of current on the AA concentration from 0.5 to 5.0 mmol L(-1).     

Probing a complex of cytochrome c and cardiolipin by magnetic circular dichroism spectroscopy: implications for the initial events in apoptosis

Oxidation of cardiolipin (CL) by its complex with cytochrome c (cyt c) plays a crucial role in triggering apoptosis. Through a combination of magnetic circular dichroism spectroscopy and potentiometric titrations, we show that both the ferric and ferrous forms of the heme group of a CL:cyt c complex exist as multiple conformers at a physiologically relevant pH of 7.4. For the ferric state, these conformers are His/Lys- and His/OH(-)-ligated. The ferrous state is predominantly high-spin and, most likely, His/-. Interconversion of the ferric and ferrous conformers is described by a single midpoint potential of -80 ± 9 mV vs SHE. These results suggest that CL oxidation in mitochondria could occur by the reaction of molecular oxygen with the ferrous CL:cyt c complex in addition to the well-described reaction of peroxides with the ferric form.     

Evaluation of immobilized redox indicators as reversible, in situ redox sensors for determining Fe(III)-reducing conditions in environmental samples

An in situ methodology based on immobilized redox indicators has been developed to determine when Fe(III)-reducing conditions exist in environmental systems. The redox indicators thionine (Thi, formal potential at pH 7 (E(7)(0')) equals 66 mV), toluidine blue O (TB, E(7)(0')=31 mV), and cresyl violet (CV, E(7)(0')=-75 mV) have been immobilized to 40-60 mum agarose beads via an amine-aldehyde coupling reaction. These beads were packed into a flow cell to allow spectrophotometric monitoring of the redox state of simple solutions and wastewater slurries pumped from in a bioreactor. Fe(II), a product of microbial activity, at levels observed in real systems reduces both the free (non-immobilized) and immobilized redox indicator to different degrees for samples with pH 6.5 or higher. At pH 7, immobilized Thi and TB are significantly reduced at Fe(II) concentrations greater than 0.1 and 0.3 mM, respectively. CV, with the lowest formal potential, requires Fe(II) levels in excess of 10 mM. The degree of reduction of the indicators (i.e. the fraction of indicator oxidized) observed during titrations can be qualitatively modeled with a simple equilibrium model based on ferrihydrite or lepidocrocite as the Fe(III)-solid phase. The reversibility of Fe(II)-indicator reactions was also demonstrated by showing that the reduced indicator becomes re-oxidized when Fe(II) levels decrease.     

Reorganization of immobilized horse and yeast cytochrome c induced by pH changes or nitric oxide binding

The redox properties of horse and yeast cytochrome c electrostatically immobilized on carboxylic acid-terminated self-assembled monolayers (SAMs) have been determined over a broad pH range (pH 3.5-8) in the absence and presence of nitric oxide. Below pH 6, both proteins exhibit comparable midpoint potentials, coverages, and electron-transfer rate constants, which suggests that they are adsorbed on the SAM in a similar fashion. Above pH 6, a sharp decrease in electron-transfer rate constants is observed for immobilized yeast cytochrome c, which is indicative of a change in the electron tunneling pathway between the heme and the electrode and hence suggests that the protein reorients on the surface. Such a decrease is not observed for horse cytochrome c and therefore must be related to the specific charge distribution on yeast cytochrome c. Apart from the charge distribution on the protein, the reorientation also seems to be related to the charge on the SAM surface. The presence of nitric oxide causes a decrease in electron-transfer rate constants of both yeast and horse cytochrome c at low pH. This is probably due to the fact that nitric oxide induces a conformational change of the protein and also changes the reorganization energy for electron transfer.     

Electrochemical reduction of NO by hemin adsorbed at pyrolitic graphite

The mechanism of the electrochemical reduction of nitric oxide (NO) by hemin adsorbed at pyrolitic graphite was investigated. The selectivity of NO reduction was probed by combining the rotating ring disk electrode (RRDE) technique with a newly developed technique called on-line electrochemical mass spectroscopy (OLEMS). These techniques show that NO reduction by adsorbed heme groups results in production of hydroxylamine (NH(2)OH) with almost 100% selectivity at low potentials. Small amounts of nitrous oxide (N(2)O) were only observed at higher potentials. The rate-determining step in NO reduction most likely consists of an electrochemical equilibrium involving a proton transfer, as can be derived from the Tafel slope value of 62 mV/dec and the pH dependence of -42 mV/pH. The almost 100% selectivity toward NH(2)OH distinguishes this system both from NO reduction on bare metal electrodes, which often yields NH(3), and from biological NO reduction in cytochrome P450nor, which yields N(2)O exclusively.     

Morphology-dependent electrochemistry and electrocatalytical activity of cytochrome c

The morphology-dependent electrochemistry and electrocatalytical activity of cytochrome c (cyt. c) were investigated at pyramidal, rodlike, and spherical gold nanostructures directly electrodeposited onto sputtered gold surfaces. Direct, reversible electron transfer of cyt. c, for the first time, was realized at nanorod-like and nanopyramidal gold surfaces without any mediators or promoters, while no redox reaction was observed at the nanospherical gold electrode. The electrochemical properties of cyt. c vary with the shape of gold nanostructures with respect to the reversibility of electrode reactions, kinetic parameters, the formal potentials (E0'), and charge-transport resistance (Rct), suggesting shape-dependent mechanisms for the electrode reactions of cyt. c. The experimental results manifest that cyt. c was stably immobilized on the nanostructured gold electrodes with different conformational changes of the heme microenvironment. Consequently, not only the electroactivity, but also the inherent biological activity of the immobilized cyt. c strongly depended on the shape of the electrode surfaces. The facilitated electron transfer combined with the intrinsic catalytical activity of cyt. c substantially constructed a third-generation H2O2 biosensor with high selectivity, quick response time, large linear range, and good sensitivity. The electrocatalytical activity of the immobilized cyt. c toward H2O2 was also found to be morphology dependent, and the linear range of H2O2 detection could be tuned by means of employing the nanostructured gold surfaces with different shapes.     

Electrochemistry of unfolded cytochrome c in neutral and acidic urea solutions

The present investigation reports the first experimental measurements of the reorganization energy of unfolded metalloprotein in urea solution. Horse heart cytochrome c (cyt c) has been found to undergo reversible one-electron transfer reactions at pH 2 in the presence of 9 M urea. In contrast, the protein is electrochemically inactive at pH 2 under low-ionic strength conditions in the absence of urea. Urea is shown to induce ligation changes at the heme iron and lead to practically complete loss of the alpha-helical content of the protein. Despite being unfolded, the electron-transfer (ET) kinetics of cyt c on a 2-mercaptoethanol-modified Ag(111) electrode remain unusually fast and diffusion controlled. Acid titration of ferric cyt c in 9 M urea down to pH 2 is accompanied by protonation of one of the axial ligands, water binding to the heme iron (pK(a) = 5.2), and a sudden protein collapse (pH < 4). The formal redox potential of the urea-unfolded six-coordinate His18-Fe(III)-H(2)O/five-coordinate His18-Fe(II) couple at pH 2 is estimated to be -0.083 V vs NHE, about 130 mV more positive than seen for bis-His-ligated urea-denatured cyt c at pH 7. The unusually fast ET kinetics are assigned to low reorganization energy of acid/urea-unfolded cyt c at pH 2 (0.41 +/- 0.01 eV), which is actually lower than that of the native cyt c at pH 7 (0.6 +/- 0.02 eV), but closer to that of native bis-His-ligated cyt b(5) (0.44 +/- 0.02 eV). The roles of electronic coupling and heme-flattening on the rate of heterogeneous ET reactions are discussed.     

Modulation of heme redox potential in the cytochrome c6 family

Cytochrome c6A is a unique dithio-cytochrome of green algae and plants. It has a very similar core structure to that of bacterial and algal cytochromes c6 but is unable to fulfill the same function of transferring electrons from cytochrome f to photosystem I. A key feature is that its heme midpoint potential is more than 200 mV below that of cytochrome c6 despite having His and Met as axial heme-iron ligands. To identify the molecular origins of the difference in potential, the structure of cytochrome c6 from the cyanobacterium Phormidium laminosum has been determined by X-ray crystallography and compared with the known structure of cytochrome c6A. One salient difference of the heme pockets is that a highly conserved Gln (Q51) in cytochrome c6 is replaced by Val (V52) in c6A. Using protein film voltammetry, we found that swapping these residues raised the c6A potential by +109 mV and decreased that of c6 by almost the same extent, -100 mV. X-ray crystallography of the V52Q protein showed that the Gln residue adopts the same configuration relative to the heme as in cytochrome c6 and we propose that this stereochemistry destabilizes the oxidized form of the heme. Consequently, replacement of Gln by Val was probably a key step in the evolution of cytochrome c6A from cytochrome c6, inhibiting reduction by the cytochrome b6f complex and facilitating establishment of a new function.     

Catalytic reduction of dioxygen and nitrite ion at a Met80Ala cytochrome c-functionalized electrode

The Met80Ala variant of yeast iso-1-cytochrome c, immobilized on a gold electrode, is found to exchange electrons efficiently with it in nondenaturing conditions and to provide robust and persistent catalytic currents for O 2 and nitrite ion reduction from pH 3 to 11. Direct covalent protein linkage to gold yields the best electrochemical and electrocatalytic performances without drastically affecting the structural properties of the bound protein compared to the freely diffusing species. Therefore, this biocatalytic interface can be of use for the amperometric detection of the above species, which are of great environmental, industrial, and clinical interest, with particular reference to the exploitation in nanostructured biosensing devices. This work shows that the use of a small engineered electron transfer (ET) protein, featuring an axial heme iron coordination position available for the binding of exogenous ligands, in place of a large heme enzyme is a viable strategy for the improvement of the heterogeneous ET rate and the stability and efficiency of sensing gold-protein interfaces over a wide range of T and pH.     

Direct electrochemistry of myoglobin based on electrodeposition of Pd nanoparticles with carbon ionic liquid electrode as basic electrode

We report on the direct electrochemistry and electrocatalytic properties of myoglobin (Mb) immobilized on a carbon ionic liquid electrode covered with a matrix composed of an ionic liquid, gellan gum, and Pd nanoparticles. UV-vis and FT-IR spectroscopy confirm that Mb retains its native structure in the composite film on the electrode. Scanning electron microscopy reveals that the nanoparticles are deposited on the surface of the Pd electrode. Cyclic voltametry gives a pair of well-defined and quasireversible redox peaks with a formal potential (E ^0′) of ?332 mV and a peak-to-peak separation of 64 mV at near-neutral pH value. The modified electrode shows good electrocatalytic activity towards the reduction of hydrogen peroxide, with a linear range between 5.0 μM and 0.27 mM and a detection limit of 1.7 μM (S/N = 3). The apparent Michaelis-Menten constant is 88 μM.

Biomaterial engineered electrodes for bioelectronics

A series of single-cysteine-containing cytochrome c, Cyt c, heme proteins including the wild-type Cyt c (from Saccharomyces cerevisiae) and the mutants (V33C, Q21C, R18C, G1C, K9C and K4C) exhibit direct electrical contact with Au-electrodes upon covalent attachment to a maleimide monolayer associated with the electrode. With the G1C-Cyt c mutant, which includes the cysteine residue in the polypeptide chain at position 1, the potential-induced switchable control of the interfacial electron transfer was observed. This heme protein includes a positively charged protein periphery that surrounds the attachment site and faces the electrode surface. Biasing of the electrode at a negative potential (-0.3 V vs. SCE) attracts the reduced Fe(II)-Cyt c heme protein to the electrode surface. Upon the application of a double-potential-step chronoamperometric signal onto the electrode, where the electrode potential is switched to +0.3 V and back to -0.3 V, the kinetics of the transient cathodic current, corresponding to the re-reduction of the Fe(III)-Cyt c, is controlled by the time interval between the oxidative and reductive potential steps. While a short time interval results in a rapid interfacial electron-transfer, ket1 = 20 s-1, long time intervals lead to a slow interfacial electron transfer to the Fe(III)-Cyt c, ket2 = 1.5 s-1. The fast interfacial electron-transfer rate-constant is attributed to the reduction of the surface-attracted Fe(III)-Cyt c. The slow interfacial electron-transfer rate constant is attributed to the electrostatic repulsion of the positively charged Cyt c from the electrode surface, resulting in long-range electron transfer exhibiting a lower rate constant. At intermediate time intervals between the oxidative and reductive steps, two populations of Cyt c, consisting of surface-attracted and surface-repelled heme proteins, are observed. Crosslinking of a layered affinity complex between the Cyt c and cytochrome oxidase, COx, on an Au-electrode yields an electrically-contacted, integrated, electrode for the four-electron reduction of O2 to water. Kinetic analysis reveals that the rate-limiting step in the bioelectrocatalytic reduction of O2 by the integrated Cyt c/COx electrode is the primary electron transfer from the electrode support to the Cyt c units.     

Oxygen activation by cytochrome p450: a thermodynamic analysis

Electrode potentials for every intermediate in the cytochrome P450 cycle were estimated and evaluated by means of an oxidation state diagram. By this approach, and within the uncertainties of the approximations, the superoxide complex of cytochrome P450 at pH 7 is oxidizing: E degrees ' (P450FeO(2)2+, H+/P450FeOOH2+) = +0.93 V, and the Gibbs energy for the reaction of the hydroperoxo complex of cytochrome P450 to form compound I and water, P450FeOOH2+ + H+ = P450FeO2+ por(*+) + H2O, is 0 kJ/mol. Although cytochrome P450FeOOH2+ and cytochrome P450FeO2+ por(*+) are approximately isoenergetic, they are likely to react at different rates with substrates and may yield different products. Homolysis of the hydroperoxo complex of cytochrome P450 to compound II and the hydroxyl radical, P450FeOOH2+ = P450FeO2+ + HO(*), is unfavorable (DeltaG degrees ' = +92 kJ/mol), as is the dissociation into HOO- and cytochrome P450Fe3+ (+73 kJ/mol). It is shown that the sum of the Gibbs energy of association for cytochrome P450Fe3+ with the hydroperoxo anion and the Gibbs energy for the one-electron reduction of cytochrome P450FeOOH2+, relative to NHE, is constant (-203 kJ/mol). While the estimated E degrees ' (P450FeO(2)2+, H+/P450FeOOH2+) = +0.93 V at pH 7 is larger than necessary to effect reduction of cytochrome P450FeO(2)2+, the magnitude of this electrode potential implies that the binding constant for cytochrome P450Fe3+ with hydrogen peroxide is ca. 3 x 106 M(-1) at pH 7. An association constant of this magnitude ensures that a fraction of cytochrome P450FeOOH2+ is available to form compound I or to react with substrates directly, while a larger one would imply that compound I is too weak an oxidant. In general, the energetics of the reduction of dioxygen to water determines the energetics of catalysis of hydroxylations by cytochrome P450. These results enable calibration of energy levels obtained for intermediates in the cytochrome P450 reaction cycle obtained by ab initio calculations and provide insights into the catalytic efficiency of cytochrome P450 and guidelines for the development of competent hydroxylation catalysts.     

离子液体和KGM修饰电极上的直接电化学

将血红蛋白固定在用室温离子液体和魔芋葡甘聚糖(KGM)水凝胶修饰的玻碳电极上,其循环伏安扫描显示一对可逆的氧化还原电流峰,克式量电位(-0.38V,vs.SCE)随溶液pH值的增大而负移,呈良好的线性关系,斜率为51 mV/pH,表明在离子液体和KGM共同修饰的电极上包埋在魔芋葡甘聚糖水凝胶中的血红蛋白发生了直接可逆的电子传递反应,并伴随有一质子的迁移过程.此外,还考察了该血红蛋白修饰电极对O2还原反应的电催化性能.     

Direct immobilization of native yeast iso-1 cytochrome C on bare gold: fast electron relay to redox enzymes and zeptomole protein-film voltammetry

Cyclic voltammetry shows that yeast iso-1-cytochrome c (YCC), chemisorbed on a bare gold electrode via Cys102, exhibits fast, reversible interfacial electron transfer (k(0) = 1.8 x 10(3) s(-1)) and retains its native functionality. Vectorially immobilized YCC relays electrons to yeast cytochrome c peroxidase, and to both cytochrome cd(1) nitrite reductase (NIR) and nitric oxide reductase from Paracoccus denitrificans, thereby revealing the mechanistic properties of these enzymes. On a microelectrode, we measured nitrite turnover by approximately 80 zmol (49 000 molecules) of NIR, coadsorbed on 0.65 amol (390 000 molecules) of YCC.