An Electrochemical Approach to Investigate Gated Electron Transfer Using a Physiological Model System:  Cytochrome <i>c</i> Immobilized on Carboxylic Acid-Terminated Alkanethiol Self-Assembled Monolayers on Gold Electrodes

173

220

A biomimetic long-range electron transfer (ET) system consisting of the blue copper protein azurin, a tunneling barrier bridge, and a gold single-crystal electrode was designed on the basis of molecular wiring self-assembly principles. This system is sufficiently stable and sensitive in a quasi-biological environment, suitable for detailed observations of long-range protein interfacial ET at the nanoscale and single-molecule levels. Because azurin is located at clearly identifiable fixed sites in well controlled orientation, the ET configuration parallels biological ET. The ET is nonadiabatic, and the rate constants display tunneling features with distance-decay factors of 0.83 and 0.91 Å ؊1 in H2O and D2O, respectively. Redoxgated tunneling resonance is observed in situ at the single-molecule level by using electrochemical scanning tunneling microscopy, exhibiting an asymmetric dependence on the redox potential. Maximum resonance appears around the equilibrium redox potential of azurin with an on͞off current ratio of Ϸ9. Simulation analyses, based on a two-step interfacial ET model for the scanning tunneling microscopy redox process, were performed and provide quantitative information for rational understanding of the ET mechanism.blue copper protein ͉ scanning tunneling microscopy ͉ nanoscale bioelectronics ͉ bioelectrochemistry C harge transfer plays key roles in many chemical and biological processes as well as in molecular electronics (1-5). For example, long-range protein electron transfer (ET) is central in aerobic respiration and photosynthesis. The importance of longrange ET is illustrated by a recent special issue of PNAS on this topic (6-12). Several articles reflect broadly the current status of this subject. However, one of the major objectives in nanoscale science and technology is to fabricate molecular electronic devices with specified functions. Molecular electronics is rooted in the concept of molecular charge transfer (particularly, molecular conductivity) (13,14). Two essential steps involved in bottom-up manipulations are (i) organizing molecules into nanoscale structures and (ii) interfacing such nanostructures with macroscopically addressable components (e.g., metal and semiconductor electrodes). Molecular electronic device function thus rests fundamentally on charge transfer through organic and͞or biological molecules and across the interface between molecules and macroscopic electrodes (15, 16). Understanding of charge transfer mechanisms has mostly been based on average results of macroscopic measurements. The advent of scanning probe microscopies, along with other supersensitive techniques, has made it possible to characterize or directly observe charge transfer through organic molecules down to the nanoscale and single-molecule levels, as illustrated by measurements of singlemolecule conductivity (17-21) and probing of molecular switching and resonant tunneling (22,23). This, however, remains a daunting challenge for proteins, with difficulties arising from the assembly of suitable st...

Section: Resultssupporting

confidence: 70%

Long-range protein electron transfer observed at the single-molecule level: In situ mapping of redox-gated tunneling resonance

Chi

Farver

Ulstrup

2005

173

220

“…Previous studies that exploited SAM-coated electrodes revealed two kinds of phenomenological behavior (17)(18)(19)(20)(21)(22)(24)(25)(26)(27)(28)(29)(30)(31). For the case of thicker SAMs (long-range ET), these studies showed an exponential decay of k et with the SAM thickness (that is a part of the ET distance, R e ); in accordance with the nonadiabatic ET (tunneling) theory (1, 3, 17-22, 24-32, 46-50):…”

supporting

confidence: 75%

Fundamental signatures of short- and long-range electron transfer for the blue copper protein azurin at Au/SAM junctions

Khoshtariya

Dolidze

Shushanyan

et al. 2010

126

The blue copper protein from Pseudomonas aeruginosa, azurin, immobilized at gold electrodes through hydrophobic interaction with alkanethiol self-assembled monolayers (SAMs) of the general type [−S − ðCH 2 Þ n − CH 3 ] (n ¼ 4, 10, and 15) was employed to gain detailed insight into the physical mechanisms of short-and long-range biomolecular electron transfer (ET). Fast scan cyclic voltammetry and a Marcus equation analysis were used to determine unimolecular standard rate constants and reorganization free energies for variable n, temperature (2-55°C), and pressure (5-150 MPa) conditions. A novel global fitting procedure was found to account for the reduced ET rate constant over almost five orders of magnitude (covering different n, temperature, and pressure) and revealed that electron exchange is a direct ET process and not conformationally gated. All the ET data, addressing SAMs with thickness variable over ca. 12 Å, could be described by using a single reorganization energy (0.3 eV), however, the values for the enthalpies and volumes of activation were found to vary with n. These data and their comparison with theory show how to discriminate between the fundamental signatures of short-and long-range biomolecular ET that are theoretically anticipated for the adiabatic and nonadiabatic ET mechanisms, respectively. electron transfer mechanism | pressure | protein friction | reorganization | temperature T he intrinsic electron transfer (ET) mechanisms of even small and otherwise well-characterized proteins such as cytochrome c or azurin (Az) are difficult to identify conclusively because of the proteins' complexity, i.e., inhomogeneous structural and dynamic properties (1-14). The use of bioelectrochemical tunneling junctions, such as self-assembled monolayer (SAM) films of variable composition and thickness on metal electrodes, with redox proteins immobilized at the solution interface (or freely diffusing to the SAM terminal groups) have been shown to provide an assembly with well-defined and variable control parameters. As such, these assemblies are well suited for fundamental studies (15-32) and offer promise for versatile nanotechnology applications (32,33). On the basis of earlier fundamental efforts, this work studies ET between a Au electrode that is coated with a SAM alkanethiol film of variable thickness and a "model" biomolecular target, the blue copper protein, Az, from Pseudomonas aeruginosa that is immobilized through hydrophobic interactions onto the SAM. As a decisive development of the preceding work (19,20,25,26), we offer unique kinetic data obtained through temperature-and pressure-variation and the mechanistic analysis through a unique global fitting procedure accounting for ET at different SAM thickness, temperature, and pressure conditions that provided the variation of the reduced ET rate constant over almost five orders of magnitude. Importantly, our previous work demonstrated that Au-deposited SAMs can withstand pressure-related stress within 5 to 150 MPa (27, 28, 34).The rate constant, k ...

“…This behavior has been attributed to a number of mechanisms: (i) dynamic modes of the solvent or protein interior that damp motion along the ET reaction coordinate (so-called ''quantum friction'') (42); (ii) electrode-derived electric field effects that influence proton rearrangements (43); and (iii) larger amplitude gating processes that produce transitions between active vs. inactive states (44)(45)(46). In the electrochemical systems, ET rates plateau in the same range that we observe for the ZnCcP:Cc complexes (200-5,000 s Ϫ1 ) (14,42,44,45). The CcP:Cc crystal systems show that leveling behavior is an intrinsic property of interfacial protein ET and not unique to electrodes.…”

Section: Structures and Redox Properties Of Znccp In Complex With Yccmentioning

confidence: 99%

Effects of interface mutations on association modes and electron-transfer rates between proteins

Kang

Crane

2005

109

Although bonding networks determine electron-transfer (ET) rates within proteins, the mechanism by which structure and dynamics influence ET across protein interfaces is not well understood. Measurements of photochemically induced ET and subsequent charge recombination between Zn-porphyrin-substituted cytochrome c peroxidase and cytochrome c in single crystals correlate reactivity with defined structures for different association modes of the redox partners. Structures and ET rates in crystals are consistent with tryptophan oxidation mediating charge recombination reactions. Conservative mutations at the interface can drastically affect how the proteins orient and dispose redox centers. Whereas some configurations are ET inactive, the wild-type complex exhibits the fastest recombination rate. Other association modes generate ET rates that do not correlate with predictions based on cofactor separations or simple bonding pathways. Inhibition of photoinduced ET at <273 K indicates gating by smallamplitude dynamics, even within the crystal. Thus, different associations achieve states of similar reactivity, and within those states conformational fluctuations enable interprotein ET.cytochrome ͉ protein dynamics ͉ protein-protein interaction ͉ electron tunneling M any long-range electron-transfer (ET) reactions in biology occur across transient protein-protein interfaces. Reaction rates depend on factors that control both electron tunneling and conformational dynamics coupled to protein association processes (1-3). As such, interprotein ET is sensitive to structure and dynamics at the interface (1, 4-11). Residue substitution (achieved either by use of protein homologs, site-directed mutants, or computations) has been a popular and powerful approach for probing how interface composition influences interprotein ET (7,(10)(11)(12)(13)(14). Nevertheless, effects of residue variation on interface structure are not often known.The natural redox partners yeast cytochrome c peroxidase (CcP) and yeast cytochrome c (yCc), whose structure as a complex was first determined in 1992 (15) and later as a covalent complex (16), have served as a paradigm for studying interprotein ET reactions (8,17). Hoffman and colleagues (8) have exploited ZnCcP substitution to photoactivate ET reactions and examine the effects of many structural and chemical perturbations on interprotein ET. In this system, the ZnCcP triplet state ( 3 ZnCcP) reduces Fe(III)Cc, and then back ET recombines the charge separation (Fig. 1). Recently, it has been demonstrated that a Trp-191 3 Phe CcP variant has much slower ET back-rates (k eb ) than wild-type (WT) CcP in the 1:1 complex with yCc (18). Thus, electron hopping through Trp-191 may accelerate the recombination reaction (Fig. 1), in analogy to the natural reaction between CcP compound I and Fe(II)Cc (19). Nevertheless, much slower ET back-rates in the complex between CcP and hCc compared with yCc, both in solution (20) and in crystals (21), indicate that ET across the protein-protein interface limits the overa...