Electron Interactions with Disulfide Bridges

Because of its electronic properties, sulfur plays a major role in a variety of metabolic processes and, more in general, in the chemistry of life. In particular, S-S bridges between cysteines are present in the amino acid backbone of proteins. Protein disulfur radical anions may decay following different paths through competing intra and intermolecular routes, including bond cleavage, disproportionation, protein-protein cross linking, and electron transfer. Indeed, mass spectrometry ECD (electron capture dissociation massspectroscopy) studies have shown that capture of low-energy (<0.2 eV) electrons by multiply protonated proteins is followed by dissociation of S-S bonds holding two peptide chains together. In view of the importance of organic sulfur chemistry, we report on electron interactions with disulphide bridges. To study these interactions we used as prototypes the molecules dimethyl sulfide [(CH3)2S] and dimethyl disulfide [(H3C)S2(CH3)]. We seek to better understand the electron-induced cleavage of the disulfide bond. To explore dissociative processes we performed electron scattering calculations with the Schwinger Multichannel Method with pseudopotentials (SMCPP), recently parallelized with OpenMP directives and optimized with subroutines for linear algebra (BLAS) and LAPACK routines. Elastic cross sections obtained for different S-S bond lengths indicate stabilization of the anion formed by electron attachment to a σ*SS antibonding orbital, such that dissociation would be expected.

Structural aspects of redox-mediated electron tunneling (Dedicated to the memory of professor Alexander M. Kuznetsov)

We investigated structural aspects of electron transfer (ET) in tunneling junctions (Au(1 1 1)vertical bar FcN vertical bar solution gap vertical bar Au STM tip) with four different redox-active N-thioalk(ano)ylferrocenes (FcN) embedded. The investigated molecules consist of a redox-active ferrocene (Fc) moiety connected via alkyl spacers with N = 4, 6, 8 and 11 carbon atoms to a thiol anchoring group. We found that for short FcNs (N = 4, 6,8) the redox-mediated ET response increases with the increase of the alkyl chain length, while no enhancement of the ET was observed for Fc1 1. The model of two-step ET with partial vibrational relaxation by Kuznetsov and Ulstrup was used to rationalize these results. The theoretical ET steps were assigned to two processes: (1) electron tunneling from the Fc group to the Au tip through the electrolyte layer and (2) electron transport from the Au(1 1 1) substrate to the Fc group through the organic adlayer. We argue that for the three short FcNs, the first process represents the rate-limiting step. The increase of the length of the alkyl chain leads to an approach of the Fc group to the STM tip, and consequently accelerates the first El' step. In case of the Fcl 1 junctions the rather high thickness of the organic layer leads to a decrease of the rate of the second ET step. In consequence, the contribution of the redox-mediated current enhancement to the total tunneling current appears to be insignificant. Our work demonstrates the importance of combined structural and transport approaches for the understanding of Er processes in electrochemical nanosystems. (C) 2010 Elsevier B.V. All rights reserved.

Integration of room temperature single electron transistor with CMOS subsystem

The single electron transistor (SET) is a charge-based device that may complement the dominant metal-oxide-semiconductor field effect transistor (MOSFET) technology. As the cost of scaling MOSFET to smaller dimensions are rising and the the basic functionality of MOSFET is encountering numerous challenges at dimensions smaller than 10nm, the SET has shown the potential to become the next generation device which operates based on the tunneling of electrons. Since the electron transfer mechanism of a SET device is based on the non-dissipative electron tunneling effect, the power consumption of a SET device is extremely low, estimated to be on the order of 10^-18J. The objectives of this research are to demonstrate technologies that would enable the mass produce of SET devices that are operational at room temperature and to integrate these devices on top of an active complementary-MOSFET (CMOS) substrate. To achieve these goals, two fabrication techniques are considered in this work. The Focus Ion Beam (FIB) technique is used to fabricate the islands and the tunnel junctions of the SET device. A Ultra-Violet (UV) light based Nano-Imprint Lithography (NIL) call Step-and-Flash- Imprint Lithography (SFIL) is used to fabricate the interconnections of the SET devices. Combining these two techniques, a full array of SET devices are fabricated on a planar substrate. Test and characterization of the SET devices has shown consistent Coulomb blockade effect, an important single electron characteristic. To realize a room temperature operational SET device that function as a logic device to work along CMOS, it is important to know the device behavior at different temperatures. Based on the theory developed for a single island SET device, a thermal analysis is carried out on the multi-island SET device and the observation of changes in Coulomb blockade effect is presented. The results show that the multi-island SET device operation highly depends on temperature. The important parameters that determine the SET operation is the effective capacitance Ceff and tunneling resistance Rt . These two parameters lead to the tunneling rate of an electron in the SET device, Γ. To obtain an accurate model for SET operation, the effects of the deviation in dimensions, the trap states in the insulation, and the background charge effect have to be taken into consideration. The theoretical and experimental evidence for these non-ideal effects are presented in this work.

An in-situ surface electrochemistry approach toward whole-cell studies: Charge transfer between Geobacter sulfurreducens and electrified metal/electrolyte interfaces through linker molecules

Electrochemical reactivity and structure properties of electrogenic bacteria, Geobacter sulfurreducens (Gs) were studied to explore the heterogeneous electron transfer at the bacteria/electrode interface using electrochemical and in-situ spectroscopic techniques. The redox behavior of Gs adsorbed on a gold electrode, which is modified with a ω-functionalized self-assembled monolayer (SAM) of alkanethiols, depends strongly on the terminal group. The latter interacts directly with outermost cytochromes embedded into the outer membrane of the Gs cells. The redox potential of bacterial cells bound electrostatically to a carboxyl-terminated SAM is close to that observed for bacteria attached to a bare gold electrode, revealing a high electronic coupling at the cell/SAM interface. The redox potentials of bacterial cells adsorbed on amino- and pyridyl-terminated SAMs are significantly different suggesting that the outermost cytochromes changes their conformation upon adsorption on these SAMs. No redox activity of Gs was found with CH3-, N(CH3)3+- and OH-terminated SAMs. Complementary in-situ spectroscopic studies on bacteria/SAMs/Au electrode assemblies were carried out to monitor structure changes of the bacterial cells upon polarization. Spectro-electrochemical techniques revealed the electrochemical turnover of the oxidized and reduced states of outer membrane cytochromes (OMCs) in Gs, providing evidence that the OMCs are responsible for the direct electron transfer to metal electrodes, such as gold or silver, during the electricity production. Furthermore, we observed spectroscopic signatures of the native structure of the OMCs and no conformational change during the oxidation/reduction process of the microorganisms. These findings indicate that the carboxyl-anchoring group provides biocompatible conditions for the outermost cytochromes of the Gs, which facilitate the heterogeneous electron transfer at the microorganism/electrode interface.

Real-time observation of the charge-transfer-to-solvent dynamics

Intermolecular electron-transfer reactions have a crucial role in biology, solution chemistry and electrochemistry. The first step of such reactions is the expulsion of the electron to the solvent, whose mechanism is determined by the structure and dynamical response of the latter. Here we visualize the electron transfer to water using ultrafast fluorescence spectroscopy with polychromatic detection from the ultraviolet to the visible region, upon photo-excitation of the so-called charge transfer to solvent states of aqueous iodide. The initial emission is short lived (~60 fs) and it relaxes to a broad distribution of lower-energy charge transfer to solvent states upon rearrangement of the solvent cage. This distribution reflects the inhomogeneous character of the solvent cage around iodide. Electron ejection occurs from the relaxed charge transfer to solvent states with lifetimes of 100–400 fs that increase with decreasing emission energy.

Proton transfer from the bulk to the bound ubiquinone QB of the reaction center in chromatophores of Rhodobacter sphaeroides: Retarded conveyance by neutral water

The mechanism of proton transfer from the bulk into the membrane protein interior was studied. The light-induced reduction of a bound ubiquinone molecule QB by the photosynthetic reaction center is accompanied by proton trapping. We used kinetic spectroscopy to measure (i) the electron transfer to QB (at 450 nm), (ii) the electrogenic proton delivery from the surface to the QB site (by electrochromic carotenoid response at 524 nm), and (iii) the disappearance of protons from the bulk solution (by pH indicators). The electron transfer to QB− and the proton-related electrogenesis proceeded with the same time constant of ≈100 μs (at pH 6.2), whereas the alkalinization in the bulk was distinctly delayed (τ ≈ 400 μs). We investigated the latter reaction as a function of the pH indicator concentration, the added pH buffers, and the temperature. The results led us to the following conclusions: (i) proton transfer from the surface-located acidic groups into the QB site followed the reduction of QB without measurable delay; (ii) the reprotonation of these surface groups by pH indicators and hydronium ions was impeded, supposedly, because of their slow diffusion in the surface water layer; and (iii) as a result, the protons were slowly donated by neutral water to refill the proton vacancies at the surface. It is conceivable that the same mechanism accounts for the delayed relaxation of the surface pH changes into the bulk observed previously with bacteriorhodopsin membranes and thylakoids. Concerning the coupling between proton pumps in bioenergetic membranes, our results imply a tendency for the transient confinement of protons at the membrane surface.

Determination of the binding sites of the proton transfer inhibitors Cd2+ and Zn2+ in bacterial reaction centers

The reaction center (RC) from Rhodobacter sphaeroides couples light-driven electron transfer to protonation of a bound quinone acceptor molecule, QB, within the RC. The binding of Cd2+ or Zn2+ has been previously shown to inhibit the rate of reduction and protonation of QB. We report here on the metal binding site, determined by x-ray diffraction at 2.5-Å resolution, obtained from RC crystals that were soaked in the presence of the metal. The structures were refined to R factors of 23% and 24% for the Cd2+ and Zn2+ complexes, respectively. Both metals bind to the same location, coordinating to Asp-H124, His-H126, and His-H128. The rate of electron transfer from QA− to QB was measured in the Cd2+-soaked crystal and found to be the same as in solution in the presence of Cd2+. In addition to the changes in the kinetics, a structural effect of Cd2+ on Glu-H173 was observed. This residue was well resolved in the x-ray structure—i.e., ordered—with Cd2+ bound to the RC, in contrast to its disordered state in the absence of Cd2+, which suggests that the mobility of Glu-H173 plays an important role in the rate of reduction of QB. The position of the Cd2+ and Zn2+ localizes the proton entry into the RC near Asp-H124, His-H126, and His-H128. Based on the location of the metal, likely pathways of proton transfer from the aqueous surface to QB⨪ are proposed.

Identification of the proton pathway in bacterial reaction centers: Replacement of Asp-M17 and Asp-L210 with Asn reduces the proton transfer rate in the presence of Cd2+

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the reduction and protonation of a bound quinone molecule QB (the secondary quinone electron acceptor). We investigated the proton transfer pathway by measuring the proton-coupled electron transfer, kAB(2) [QA⨪QB⨪ + H+ → QA(QBH)−] in native and mutant RCs in the absence and presence of Cd2+. Previous work has shown that the binding of Cd2+ decreases kAB(2) in native RCs ≈100-fold. The preceding paper shows that bound Cd2+ binds to Asp-H124, His-H126, and His-H128. This region represents the entry point for protons. In this work we investigated the proton transfer pathway connecting the entry point with QB⨪ by searching for mutations that greatly affect kAB(2) (≳10-fold) in the presence of Cd2+, where kAB(2) is limited by the proton transfer rate (kH). Upon mutation of Asp-L210 or Asp-M17 to Asn, kH decreased from ≈60 s−1 to ≈7 s−1, which shows the important role that Asp-L210 and Asp-M17 play in the proton transfer chain. By comparing the rate of proton transfer in the mutants (kH ≈ 7 s−1) with that in native RCs in the absence of Cd2+ (kH ≥ 104 s−1), we conclude that alternate proton transfer pathways, which have been postulated, are at least 103-fold less effective.

Identification of the proton pathway in bacterial reaction centers: Inhibition of proton transfer by binding of Zn2+ or Cd2+

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the light induced two-electron, two-proton reduction of a bound quinone molecule QB (the secondary quinone acceptor). A unique pathway for proton transfer to the QB site had so far not been determined. To study the molecular basis for proton transfer, we investigated the effects of exogenous metal ion binding on the kinetics of the proton-assisted electron transfer kAB(2) (QA−•QB−• + H+ → QA(QBH)−, where QA is the primary quinone acceptor). Zn2+ and Cd2+ bound stoichiometrically to the RC (KD ≤ 0.5 μM) and reduced the observed value of kAB(2) 10-fold and 20-fold (pH 8.0), respectively. The bound metal changed the mechanism of the kAB(2) reaction. In native RCs, kAB(2) was previously shown to be rate-limited by electron transfer based on the dependence of kAB(2) on the driving force for electron transfer. Upon addition of Zn2+ or Cd2+, kAB(2) became approximately independent of the electron driving force, implying that the rate of proton transfer was reduced (≥ 102-fold) and has become the rate-limiting step. The lack of an effect of the metal binding on the charge recombination reaction D+•QAQB−• → DQAQB suggests that the binding site is located far (>10 Å) from QB. This hypothesis is confirmed by preliminary x-ray structure analysis. The large change in the rate of proton transfer caused by the stoichiometric binding of the metal ion shows that there is one dominant site of proton entry into the RC from which proton transfer to QB−• occurs.

Electron tunneling in protein crystals

The current understanding of electron tunneling through proteins has come from work on systems where donors and acceptors are held at fixed distances and orientations. The factors that control electron flow between proteins are less well understood, owing to uncertainties in the relative orientations and structures of the reactants during the very short time that tunneling occurs. As we report here, the way around such structural ambiguity is to examine oxidation–reduction reactions in protein crystals. Accordingly, we have measured and analyzed the kinetics of electron transfer between native and Zn-substituted tuna cytochrome c (cyt c) molecules in crystals of known structure. Electron transfer rates [(320 s−1 for *Zn-cyt c → Fe(III)-cyt c; 2000 s−1 for Fe(II)-cyt c → Zn-cyt c+)] over a Zn–Fe distance of 24.1 Å closely match those for intraprotein electron tunneling over similar donor–acceptor separations. Our results indicate that van der Waals interactions and water-mediated hydrogen bonds are effective coupling elements for tunneling across a protein–protein interface.

On the role of the K-proton transfer pathway in cytochrome c oxidase

Cytochrome c oxidase is a membrane-bound enzyme that catalyzes the four-electron reduction of oxygen to water. This highly exergonic reaction drives proton pumping across the membrane. One of the key questions associated with the function of cytochrome c oxidase is how the transfer of electrons and protons is coupled and how proton transfer is controlled by the enzyme. In this study we focus on the function of one of the proton transfer pathways of the R. sphaeroides enzyme, the so-called K-proton transfer pathway (containing a highly conserved Lys(I-362) residue), leading from the protein surface to the catalytic site. We have investigated the kinetics of the reaction of the reduced enzyme with oxygen in mutants of the enzyme in which a residue [Ser(I-299)] near the entry point of the pathway was modified with the use of site-directed mutagenesis. The results show that during the initial steps of oxygen reduction, electron transfer to the catalytic site (to form the “peroxy” state, Pr) requires charge compensation through the proton pathway, but no proton uptake from the bulk solution. The charge compensation is proposed to involve a movement of the K(I-362) side chain toward the binuclear center. Thus, in contrast to what has been assumed previously, the results indicate that the K-pathway is used during oxygen reduction and that K(I-362) is charged at pH ≈ 7.5. The movement of the Lys is proposed to regulate proton transfer by “shutting off” the protonic connectivity through the K-pathway after initiation of the O2 reduction chemistry. This “shutoff” prevents a short-circuit of the proton-pumping machinery of the enzyme during the subsequent reaction steps.

Association of glycolate oxidation with photosynthetic electron transport in plant and algal chloroplasts.

Photosynthetic carbon metabolism is initiated by ribulose-bisphosphate carboxylase/oxygenase (Rubisco), which uses both CO2 and O2 as substrates. One 2-phosphoglycolate (P-glycolate) molecule is produced for each O2 molecule fixed. P-glycolate has been considered to be metabolized exclusively via the oxidative photosynthetic carbon cycle. This paper reports an additional pathway for P-glycolate and glycolate metabolism in the chloroplasts. Light-dependent glycolate or P-glycolate oxidation by osmotically shocked chloroplasts from the algae Dunaliella or spinach leaves was measured by three electron acceptors, methyl viologen (MV), potassium ferricyanide, or dichloroindophenol. Glycolate oxidation was assayed with 3-(3,4)-dichlorophenyl)-1,1-dimethylurea (DCMU) as oxygen uptake in the presence of MV at a rate of 9 mol per mg of chlorophyll per h. Washed thylakoids from spinach leaves oxidized glycolate at a rate of 22 mol per mg of chlorophyll per h. This light-dependent oxidation was inhibited completely by SHAM, an inhibitor of quinone oxidoreductase, and 75% by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which inhibits electron transfer from plastoquinone to the cytochrome b6f complex. SHAM stimulated severalfold glycolate excretion by algal cells, Dunaliella or Chlamydomonas, and by isolated Dunaliella chloroplasts. Glycolate and P-glycolate were oxidized about equally well to glyoxylate and phosphate. On the basis of results of inhibitor action, the possible site which accepts electrons from glycolate or P-glycolate is a quinone after the DCMU site but before the DBMIB site. This glycolate oxidation is a light-dependent, SHAM-sensitive, glycolate-quinone oxidoreductase system that is associated with photosynthetic electron transport in the chloroplasts.

Potentiation of proton transfer function by electrostatic interactions in photosynthetic reaction centers from Rhodobacter sphaeroides: First results from site-directed mutation of the H subunit.

The x-ray crystallographic structure of the photosynthetic reaction center (RC) has proven critical in understanding biological electron transfer processes. By contrast, understanding of intraprotein proton transfer is easily lost in the immense richness of the details. In the RC of Rhodobacter (Rb.) sphaeroides, the secondary quinone (QB) is surrounded by amino acid residues of the L subunit and some buried water molecules, with M- and H-subunit residues also close by. The effects of site-directed mutagenesis upon RC turnover and quinone function have implicated several L-subunit residues in proton delivery to QB, although some species differences exist. In wild-type Rb. sphaeroides, Glu L212 and Asp L213 represent an inner shell of residues of particular importance in proton transfer to QB. Asp L213 is crucial for delivery of the first proton, coupled to transfer of the second electron, while Glu L212, possibly together with Asp L213, is necessary for delivery of the second proton, after the second electron transfer. We report here the first study, by site-directed mutagenesis, of the role of the H subunit in QB function. Glu H173, one of a cluster of strongly interacting residues near QB, including Asp L213, was altered to Gln. In isolated mutant RCs, the kinetics of the first electron transfer, leading to formation of the semiquinone, QB-, and the proton-linked second electron transfer, leading to the formation of fully reduced quinol, were both greatly retarded, as observed previously in the Asp L213 --> Asn mutant. However, the first electron transfer equilibrium, QA-QB <==> QAQB-, was decreased, which is opposite to the effect of the Asp L213 --> Asn mutation. These major disruptions of events coupled to proton delivery to QB were largely reversed by the addition of azide (N3-). The results support a major role for electrostatic interactions between charged groups in determining the protonation state of certain entities, thereby controlling the rate of the second electron transfer. It is suggested that the essential electrostatic effect may be to "potentiate" proton transfer activity by raising the pK of functional entities that actually transfer protons in a coupled fashion with the second electron transfer. Candidates include buried water (H3O+) and Ser L223 (serine-OH2+), which is very close to the O5 carbonyl of the quinone.

Mechanistic Importance of Redox Potentials and Conformational Flexibility in Electron-Transferring Flavoproteins

The mitochondrial matrix flavoproteins electron transfer flavoprotein (ETF) and electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) are responsible for linking fatty acid β-oxidation with the main mitochondrial respiratory chain. Electrons derived from flavoprotein dehydrogenases are transferred sequentially through ETF and ETF-QO to ubiquinone and then into the respiratory chain via complex III. In this study, the effects of changes in ETF-QO redox potentials on its activity and the conformational flexibility of ETF were investigated. ETF-QO contains one [4Fe-4S]2+,1+ and one flavin adenine dinucleotide (FAD). In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. FAD redox potentials were measured by potentiometric titration probed by electron paramagnetic resonance (EPR) spectroscopy. The N338T and N338A mutations lowered the midpoint potentials, which resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF1e- catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone, but not in electron transfer from ETF to ETF-QO. Therefore it is proposed that the iron-sulfur cluster is the immediate acceptor from ETF. It has been proposed that the αII domain of ETF is mobile, allowing promiscuous interactions with structurally different partners. Double electron-electron resonance (DEER) was used to measure the distance between spin labels at various sites and an enzymatically reduced FAD cofactor in Paracoccus denitrificans ETF. Two or three interspin distance distributions were observed for spin-labels in the αI (A43C) and βIII (A111C) domains, but only one is observed for a label in the βII (A210C) domain. This suggests that the αII domain adopts several stable conformations which may correspond to a closed/inactive conformation and an open/active conformation. An additional mutation, E162A, was introduced to increase the mobility of the αII domain. The E162A mutation doubled the activity compared to wild-type and caused the distance distributions to become wider. The DEER method has the potential to characterize conformational changes in ETF that occur when it interacts with various redox partners.

Time-resolved spectroscopy of the metal-to-metal charge transfer excited state in dinuclear cyano-bridged mixed-valence complexes

Visible pump-probe spectroscopy has been used to identify and characterize short-lived metal-to-metal charge transfer (MMCT) excited states in a group of cyano-bridged mixed-valence complexes of the formula [(LCoNCMII)-N-III(CN)(5)](-), where L is a pentadentate macrocyclic pentaamine (L-14) or triamine-dithiaether (L-14S) and M is Fe or Ru. Nanosecond pump-probe spectroscopy on frozen solutions of [(LCoNCFeII)-Co-14-N-III(CN)(5)](-) and [(LCoNCFeII)-Co-14S-N-III(CN)(5)](-) at 11 K enabled the construction of difference transient absorption spectra that featured a rise in absorbance in the region of 350-400 nm consistent with the generation of the ferricyanide chromophore of the photoexcited complex. The MMCT excited state of the Ru analogue [(LCoNCRuII)-Co-14-N-III(CN)(5)](-) was too short-lived to allow its detection. Femtosecond pump-probe spectroscopy on aqueous solutions of [(LCoNCFeII)-Co-14-N-III(CN)(5)](-) and [(LCoNCFeII)-Co-14S-N-III(CN)(5)](-) at room temperature enabled the lifetimes of their Co-II-Fe-III MMCT excited states to be determined as 0.8 and 1.3 ps, respectively.