Biomedical subjects

Peter Hildebrandt

Publications and source records attributed to Peter Hildebrandt.

At least 19 recordsLinked to original sources

PubMed2006

Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation.

The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pK(a) from 11.1 (wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild-type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photo-induced protein structural changes that in the wild-type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc --> Pfr transition and most likely coupled to the transient proton re-uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.

Agrobacterium tumefaciens↗

PubMed2006

Time-resolved methods in Biophysics. 1. A novel pump and probe surface-enhanced resonance Raman approach for studying biological photoreceptors.

This article describes a method, based on surface-enhanced resonance Raman (SERR) spectroscopy, for studying the reaction dynamics of photoreceptors immobilized on metal electrodes. Time resolution and fresh sample conditions are achieved by synchronizing the rotational and translational motion of a novel kinematic electrode with the duration and time delay between the pump and probe events. The power and sensitivity of the method is illustrated by studying the photocycle of the sensory photoreceptor NpSRII and its sensitivity to the applied electric field. The results are compared with time-resolved resonance Raman measurements in solution.

Biophysics↗

PubMed2006

Bias from H2 cleavage to production and coordination changes at the Ni-Fe active site in the NAD+-reducing hydrogenase from Ralstonia eutropha.

The soluble NAD+-reducing Ni-Fe hydrogenase (SH) from Ralstonia eutropha H16 is remarkable because it cleaves hydrogen in the presence of dioxygen at a unique Ni-Fe active site (Burgdorf et al. (2005) J. Am. Chem. Soc. 127, 576). By X-ray absorption (XAS), FTIR, and EPR spectroscopy, we monitored the structure and oxidation state of its metal centers during H2 turnover. In NADH-activated protein, a change occurred from the (CN)O2Ni(II)(mu-S)2Fe(II)(CN)3(CO) site dominant in the wild-type SH to a standard-like S2Ni(II)(mu-S)2Fe(II)(CN)2(CO) site as the prevailing species in a specific mutant protein, HoxH-H16L. The wild-type SH primarily was active in H2 cleavage. The nonstandard reaction mechanism does not involve stable EPR-detectable trivalent Ni oxidation states, namely, the Ni-A,B,C states as observed in standard hydrogenases. In the HoxH-mutant protein H16L, H2 oxidation was impaired, but H2 production occurred via a stable Ni-C state (Ni(III)-H(-)-Fe(II)), suggesting a reaction sequence similar to that of standard hydrogenases. It is proposed that reductive activation by NADH of both wild-type and H16L proteins causes the release of an oxygen species from Ni and is initiated by electron transfer from a [2Fe-2S] cluster in the HoxU subunit that at first becomes reduced by electrons from NADH. Electrons derived from H2 cleavage, on the other hand, are transferred to NAD+ via a different pathway involving a [4Fe-4S] cluster in HoxY, which is reducible only in wild-type SH but not in the H16L variant.

Binding Sites↗

PubMed2006

Calculation of vibrational spectra of linear tetrapyrroles. 4. Methine bridge C-H out-of-plane modes.

Quantum chemical force fields obtained by density functional theory (DFT) calculations systematically overestimate the frequencies of normal modes including ethylenic C-H out-of-plane (HOOP) coordinates. Compensation of this deviation requires a specific scaling factor for this type of coordinate that is distinctly lower than those applicable to out-of-plane coordinates in general. Such a specific scaling factor (0.900) has been optimized for the DFT(B3LYP) level of theory on the basis of vibrational analyses of training molecules including the HOOP coordinate. Thus, the root-mean-square deviation for the calculated frequencies of these modes is reduced from 16 to 8 cm(-1). Although Raman intensities are yet not reproduced in a satisfactory manner, implementation of the HOOP scaling factor into the set of global scaling factors determined previously (Magdo et al. J. Phys. Chem. A 1999, 103, 289-303) allows for a substantially improved reproduction of the experimental (resonance) Raman spectra of test molecules including linear methine-bridged tetrapyrroles. A very good agreement between calculated and experimental spectra is noted for the phycocyanobilin dimethylester dimer as well as for the protein-bound phycocyanobilin in the antenna pigment alpha-CPC. However, for the phycocyanobilin chromophore in the P(r) state of the plant photoreceptor phytochrome phyA, considerable deviations remain in the spectral range between 800 and 500 cm(-1), which are attributed to the effect of specific protein-chromophore interactions. The influence of the protein environment is not considered in the present calculations that refer to the molecule in vacuo.

Models, Chemical↗

PubMed2006

Heme coordination states of unfolded ferrous cytochrome C.

The structural changes of ferrous Cyt-c that are induced by binding to SDS micelles, phospholipid vesicles, DeTAB, and GuHCl as well as by high temperatures and changes in the pH have been studied by RR and UV-Vis absorption spectroscopies. Four species have been identified in which the native methionine-80 ligand is removed from the heme iron. This coordination site is either occupied by a histidine (His-33 or His-26) to form a 6cLS configuration, which is the prevailing species in GuHCl at pH 7.0 and ambient temperature, or remains vacant to yield a 5cHS configuration. The three identified 5cHS species differ with respect to the hydrogen-bond interactions of the proximal histidine ligand (His-18) and include a nonhydrogen-bonded, a hydrogen-bonded, and a deprotonated imidazole ring. These structural motifs have been found irrespective of the unfolding conditions used. An unambiguous spectroscopic distinction of these 5cHS species is possible on the basis of the Fe-N(imidazole) stretching vibrations, the RR bands in the region between 1300 and 1650 cm(-1), and the electronic transitions in the Soret- and Q-band regions. In acid and neutral solutions, the species with a hydrogen-bonded and a nonhydrogen-bonded His-18 prevail, whereas in alkaline solutions a configuration with a deprotonated His-18 ligand is also observed. Upon lowering the pH or increasing the temperature in GuHCl solutions, the structure on the proximal side of the heme is perturbed, resulting in a loss of the hydrogen-bond interactions of the His-18 ligand. Conversely, the hydrogen-bonded His-18 of ferrous Cyt-c is stabilized by electrostatic interactions which increase in strength from phospholipid vesicles to SDS micelles. The results here suggest that unfolding of Cyt-c is initiated by the rupture of the Fe-Met-80 bond and structural reorganizations on the distal side of the heme pocket, whereas the proximal part is only affected in a later stage of the denaturation process.

Animals↗

PubMed2006

Voltammetry and in situ scanning tunnelling microscopy of de novo designed heme protein monolayers on Au(111)-electrode surfaces.

In the present work, we report the electrochemical characterization and in situ scanning tunnelling microscopy (STM) studies of monolayers of an artificial de novo designed heme protein MOP-C, covalently immobilized on modified Au(111) surfaces. The protein forms closely packed monolayers, which remain electroactive upon immobilization. In situ STM images show circular structures indicating that MOP-C stands upright on the surface in accordance with the molecular design. Despite the large spatial extension of MOP-C, about 5 nm in height, conditions could be found where tip/sample interaction is minimal and proteins could be imaged without detectable tip interference. The results indicate further that the structural sensitivity of (in situ) STM depends to a significant extent on associated electron transfer kinetics. In the present case, the heme group does not contribute significantly to the tunnelling current, apparently due to slow electron transfer kinetics. As a consequence, STM images of heme-containing and heme-free MOP-C did not reveal any notable differences in apparent height or physical extension. The apparent height of heme-containing MOP-C did not show any dependence on the substrate potential being varied around the redox potential of the protein. The mere presence of an accessible molecular energy level is not sufficient to result in detectable tunnelling current modulation.

Adsorption↗

PubMed2006

Redox processes of cytochrome c immobilized on solid supported polyelectrolyte multilayers.

The heme protein cytochrome c (Cyt-c), immobilized on polyelectrolyte multilayers on a silver electrode, was studied by stationary and time-resolved surface-enhanced resonance Raman (SERR) spectroscopy to probe the redox site structure and the mechanism and dynamics of the potential-dependent interfacial processes. The layers were built up by sequential adsorption of polycations (poly[ethylene imine] (PEI); polyallylamine hydrochloride (PAH)) and polyanions (poly[styrene sulfonate] (PSS)). All multilayers terminated by PSS electrostatically bind Cyt-c. On PEI/PSS coatings, Cyt-c is peripherally bound and fully redox-active. Due to the interfacial potential drop, the apparent redox potential is lowered by 40 mV compared to that in solution. The rate constant for the heterogeneous electron transfer (ET) of ca. 0.1 s(-1) is consistent with electron tunneling through largely ordered PEI/PSS layers. ET is coupled to a reversible conformational transition of Cyt-c that involves a change of the coordination pattern of the heme. Additional (PAH/PSS) double layers cause a broadening of the redox transition and a drastic negative shift of the redox potential, which is attributed to the formation of PSS/Cyt-c complexes. It is concluded that Cyt-c can effectively compete with PAH for binding of PSS, resulting in a rearrangement of the layered structure and a penetration of the PSS-bound Cyt-c into the PAH/PSS double layers. This conclusion is consistent with SERR intensity and quartz microbalance measurements. ET was found to be overpotential-independent and faster than that for PEI/PSS coatings, which is interpreted in terms of specific PSS/Cyt-c complexes serving as gates for the heterogeneous ET.

Cytochromes c↗

PubMed2005

Long distance electron transfer in cytochrome c oxidase immobilised on electrodes. A surface enhanced resonance Raman spectroscopic study.

Cytochrome c oxidase was tethered to a functionalised Ag electrode via a histidine-tag on the C-terminus of subunit I or II and embedded in a phospholipid bilayer. The uniformly oriented membrane-bound proteins were studied by surface enhanced resonance Raman spectroscopy (SERRS) that reveals preservation of the native structures of the heme a and heme a(3) sites. On the basis of time-dependent SERRS measurements, the rate constant for the heterogeneous electron transfer to heme a was determined to be 0.002 s(-1) independent of the enzyme orientation and the overpotential. Taking into account that the electrode-to-heme a distance is larger than 50 A, these findings suggest an electron hopping mechanism in which the Cu(A) center is not involved. Electrochemical reduction is restricted to heme a whereas electron transfer from heme a to heme a(3), which in solution occurs on the nanosecond time scale, is drastically slowed down. It may be that the network of cooperativities that links intramolecular electron transfer and proton translocation is perturbed in the immobilised enzyme, possibly due to the effect of the interfacial electric field.

Chemical Phenomena↗

PubMed2005

Conformational transitions and redox potential shifts of cytochrome P450 induced by immobilization.

Cytochrome P450 (P450) from Pseudomonas putida was immobilized on Ag electrodes coated with self-assembled monolayers (SAMs) via electrostatic and hydrophobic interactions as well as by covalent cross-linking. The redox and conformational equilibria of the immobilized protein were studied by potential-dependent surface-enhanced resonance Raman spectroscopy. All immobilization conditions lead to the formation of the cytochrome P420 (P420) form of the enzyme. The redox potential of the electrostatically adsorbed P420 is significantly more positive than in solution and shows a steady downshift upon shortening of the length of the carboxyl-terminated SAMs, i.e., upon increasing the strength of the local electric field. Thus, two opposing effects modulate the redox potential of the adsorbed enzyme. First, the increased hydrophobicity of the heme environment brought about by immobilization on the SAM tends to upshift the redox potential by stabilizing the formally neutral ferrous form. Second, increasing electric fields tend to stabilize the positively charged ferric form, producing the opposite effect. The results provide insight into the parameters that control the structure and redox properties of heme proteins and contribute to the understanding of the apparently anomalous behavior of P450 enzymes in bioelectronic devices.

Biosensing Techniques↗

PubMed2005

Midpoint potentials of hemes a and a3 in the quinol oxidase from Acidianus ambivalens are inverted.

The aa3 type B oxygen reductase from the thermophilic archaeon Acidianus ambivalens (QO) was immobilized on silver electrodes and studied by potential-dependent surface-enhanced resonance Raman (SERR) spectroscopy. The immobilized enzyme retains the native structure at the level of the heme pockets and exhibits reversible electrochemistry. From the potential dependence of specific spectral marker bands, the midpoint potentials of hemes a and a3 were unambiguously determined for the first time, being 320 +/- 20 mV for the former and 390 +/- 20 mV for the latter. Both hemes could be treated as independent one-electron Nernstian redox couples, indicating that the interaction potential is smaller than 50 mV. The reversed order of the midpoint potentials compared to those of type A (mitochondrial-like) oxidases, as well as the lack of substantial Coulombic interactions, suggests a different mechanism of electroprotonic energy transduction. In contrast to type A enzymes, a-a3 intraprotein electron transfer in QO is already guaranteed by the order of the midpoint potentials at the onset of enzyme reduction and, therefore, does not require a complex network of cooperativities to ensure exergonicity. In the immobilized state, conformational transitions of the QO a3-CuB active site, which are believed to be essential for proton translocation, are drastically slowed compared to those in solution. We ascribe this finding to the effect of the interfacial electric field, which is of the same order of magnitude as in biological membranes. These results suggest that the membrane potential may play an active role in the regulation of the enzymatic activity of QO.

Archaea↗

PubMed2005

Redox and redox-coupled processes of heme proteins and enzymes at electrochemical interfaces.

Modern bioelectrochemical methods rely upon the immobilisation of redox proteins and enzymes on electrodes coated with biocompatible materials to prevent denaturation. However, even when protein denaturation is effectively avoided, heterogeneous protein electron transfer is often coupled to non-Faradaic processes like reorientation, conformational transitions or acid-base equilibria. Disentangling these processes requires methods capable of probing simultaneously the structure and reaction dynamics of the adsorbed species. Here we provide an overview of the recent developments in Raman and infrared surface-enhanced spectroelectrochemical techniques applied to the study of soluble and membrane bound redox heme proteins and enzymes. Possible biological implications of the findings are critically discussed.

Biosensing Techniques↗

PubMed2005

Active sites of two orthologous cytochromes P450 2E1: differences revealed by spectroscopic methods.

Cytochromes P450 2E1 of human and minipig origin were examined by absorption spectroscopy under high hydrostatic pressure and by resonance Raman spectroscopy. Human enzyme tends to denature to the P420 form more easily than the minipig form; moreover, the apparent compressibility of the heme active site (as judged from a redshift of the absorption maximum with pressure) is greater than that of the minipig counterpart. Relative compactness of the minipig enzyme is also seen in the Raman spectra, where the presence of planar heme conformation was inferred from band positions characteristic of the low-spin heme with high degree of symmetry. In this respect, the CYP2E1 seems to be another example of P450 conformational heterogeneity as shown, e.g., by Davydov et al. for CYP3A4 [Biochem. Biophys. Res. Commun. 312 (2003) 121-130]. The results indicate that the flexibility of the CYP active site is likely one of its basic structural characteristics.

Animals↗

PubMed2005

The structure of the Ni-Fe site in the isolated HoxC subunit of the hydrogen-sensing hydrogenase from Ralstonia eutropha.

The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha which forms a [HoxBC]2 complex functions as a hydrogen sensor under aerobic conditions. We have studied a novel Strep-tag isolate of the RH large subunit, HoxC(ST), which lacks the Fe-S clusters of HoxB, allowing for structure determination of the catalytic site by X-ray absorption spectroscopy both at the Ni and, for the first time, also at the Fe K-edge. This technique, together with Fourier-transform infrared spectroscopy, revealed a Ni-Fe site with [O1(CysS)2Ni(II)(mu-SCys)2Fe(II)(CN)2(CO)] structure in about 50% of HoxC(ST) and a [(CysS)2Fe(II)(CN)2(CO)] site lacking Ni in the remainder protein. Possibly both sites may be intermediates in the maturation process of the RH.

Bacterial Proteins↗

PubMed2005

Light-induced proton release of phytochrome is coupled to the transient deprotonation of the tetrapyrrole chromophore.

The Pr --> Pfr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-R(C) were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in Pr and ZZEssa in Pfr, with all pyrrole nitrogens being protonated. Photoexcitation of Pr is followed by (at least) three thermal relaxation components in the formation of Pfr with time constants of 230 micros and 3.1 and 260 ms. H2O/D2O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-R(C), respectively. Resonance Raman measurements of Meta-R(C) indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-R(C) are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the Pr --> Pfr photocon-version mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the Pfr formation that switch between the active and the inactive state of the photoreceptor.

Agrobacterium tumefaciens↗

PubMed2005

Trinuclear nickel complexes with triplesalen ligands: simultaneous occurrence of mixed valence and valence tautomerism in the oxidized species.

The coordination chemistries of the triple tetradentate triplesalen ligands H(6)talen, H(6)talen(t)(-)(Bu)(2), and H(6)talen(NO)(2) have been investigated with nickel(II). These triplesalen ligands provide three salen-like coordination environments bridged in a meta-phenylene arrangement by a phloroglucinol backbone. The structures of the complexes [(talen)Ni(II)(3)], [(talen(t)(-)(Bu)(2)Ni(II)(3)], and [(talen(NO)(2)Ni(II)(3)] have been determined by single-crystal X-ray diffraction. All three compounds are composed of neutral trinuclear complexes with square-planar coordinated Ni(II) ions in a salen-like coordination environment. Whereas the overall molecular structure of [(talen(NO)(2)Ni(II)(3)] is nearly planar, the structures of [(talen)Ni(II)(3)] and [(talen(t)(-)(Bu)(2)Ni(II)(3)] are bowl-shaped as a result of ligand folding. The strongest ligand folding occurs at the central nickel-phenolate bond of [(talen(t)(-)(Bu)(2)Ni(II)(3)], resulting in the formation of a chiral hemispherical pocket. The dependence of the physical properties by the substituents on the terminal phenolates has been studied by FTIR, resonance Raman, UV-vis-NIR absorption, and electrochemistry. The three nickel-salen subunits are electronically interacting via the pi system of the bridging phloroglucinol backbone. The strength of this interaction is mediated by two opposing effects: the electron density at the terminal phenolates and the folding of the ligand at the central phenolates. The parent complex [(talen)Ni(II)(3)] is irreversibly oxidized at 0.32 V versus ferrocenium/ferrocene (Fc(+)/Fc), whereas [(talen(t)(-)(Bu)2)Ni(II)(3)] and [(talen(NO)(2)Ni(II)(3)] exhibit reversible oxidations at 0.22 V versus Fc(+)/Fc and 0.52 V versus Fc(+)/Fc, respectively. The oxidized species [(talen(t)(-)(Bu)(2)Ni(3)](+) and [(talen(NO)(2)Ni(3)](+) undergo a valence-tautomeric transformation involving a Ni(III) and a phenoxyl radical species, as observed by EPR spectroscopy. Thus, these oxidized forms exhibit the phenomena of valence tautomerism and mixed valence simultaneously. The extent of delocalization of the radical species and of the Ni(III) species is discussed.

Crystallography, X-Ray↗

PubMed2005

Calculation of vibrational spectra of linear tetrapyrroles. 3. Hydrogen-bonded hexamethylpyrromethene dimers.

The structure and vibrational spectra of hexamethylpyrromethene (HMPM) have been investigated by X-ray crystallography, IR and Raman spectroscopies, and density functional theory calculations. HMPM crystallizes in the form of dimers, which are held together by bifurcated N-H(...N)(2) hydrogen bonds, involving one intramolecular and one intermolecular N-H...N interaction. The monomers are essentially planar, and the mean planes of the monomers lie approximately perpendicular to one another, so that the four N atoms in the dimer form a distorted tetrahedron. The structure of the HMPM dimer is well-reproduced by B3LYP/6-31G calculations. A comparison of the calculated geometry of the dimer with that of the monomer reveals only small changes in the N-H...N entity and the methine bridge angles upon dimerization. These are a result of weakening of the intramolecular N-H...N hydrogen bond and the formation of a more linear N-H...N intermolecular hydrogen bond. Using an empirical relation between the shift of the N-H stretching frequency of pyrrole and the enthalpy of adduct formation with bases [Nozari, M. S.; Drago, R. S. J. Am. Chem. Soc. 1970, 92, 7086-7090], estimates of the strength of the intra- and intermolecular hydrogen bonds are obtained. IR and Raman spectroscopies of HMPM and its isotopomers deuterated at the pyrrolic nitrogen atom and at the methine bridge reveal that the molecule is monomeric in nonpolar organic solvents but dimeric in a solid Ar matrix and in KBr pellets. The matrix IR spectra show a splitting of vibrational modes for the dimer, particularly those involving the N-H coordinates. Due to intrinsic deficiencies of the B3LYP/6-31G approximation, a satisfactory reproduction of these modes of the monomeric and dimeric HMPM requires specific adjustments of the NH scaling factors for the calculated force constants and, in the case of the NH out-of-plane modes of HMPM dimers, also of intra- and intermolecular coupling constants. This parametrization does not significantly affect the other calculated modes, which in general reveal a very good agreement with the experimental data.

Journal Article↗

PubMed2005

Reduction of unusual iron-sulfur clusters in the H2-sensing regulatory Ni-Fe hydrogenase from Ralstonia eutropha H16.

The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha functions as a hydrogen sensor. The RH consists of the large subunit HoxC housing the Ni-Fe active site and the small subunit HoxB containing Fe-S clusters. The heterolytic cleavage of H(2) at the Ni-Fe active site leads to the EPR-detectable Ni-C state of the protein. For the first time, the simultaneous but EPR-invisible reduction of Fe-S clusters during Ni-C state formation was demonstrated by changes in the UV-visible absorption spectrum as well as by shifts of the iron K-edge from x-ray absorption spectroscopy in the wild-type double dimeric RH(WT) [HoxBC](2) and in a monodimeric derivative designated RH(stop) lacking the C-terminal 55 amino acids of HoxB. According to the analysis of iron EXAFS spectra, the Fe-S clusters of HoxB pronouncedly differ from the three Fe-S clusters in the small subunits of crystallized standard Ni-Fe hydrogenases. Each HoxBC unit of RH(WT) seems to harbor two [2Fe-2S] clusters in addition to a 4Fe species, which may be a [4Fe-3S-3O] cluster. The additional 4Fe-cluster was absent in RH(stop). Reduction of Fe-S clusters in the hydrogen sensor RH may be a first step in the signal transduction chain, which involves complex formation between [HoxBC](2) and tetrameric HoxJ protein, leading to the expression of the energy converting Ni-Fe hydrogenases in R. eutropha.

Amino Acid Sequence↗

PubMed2005

Electric-field-induced redox potential shifts of tetraheme cytochromes c3 immobilized on self-assembled monolayers: surface-enhanced resonance Raman spectroscopy and simulation studies.

The tetraheme protein cytochrome c(3) (Cyt-c(3)) from Desulfovibrio gigas, immobilized on a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid, is studied by theoretical and spectroscopic methods. Molecular dynamics simulations indicate that the protein docks to the negatively charged SAM via its lysine-rich domain around the exposed heme IV. Complex formation is associated with only little protein structural perturbations. This finding is in line with the resonance Raman and surface-enhanced resonance Raman (SERR) spectroscopic results that indicate essentially the same heme pocket structures for the protein in solution and adsorbed on SAM-coated Ag electrodes. Electron- and proton-binding equilibrium calculations reveal substantial negative shifts of the redox potentials compared to the protein in solution. The magnitude of these shifts decreases in the order heme IV (-161 mV) > heme III (-73 mV) > heme II (-57 mV) > heme I (-26 mV), resulting in a change of the order of reduction. These shifts originate from the distance-dependent electrostatic interactions between the SAM headgroups and the individual hemes, leading to a stabilization of the oxidized forms. The results of the potential-dependent SERR spectroscopic analyses are consistent with the theoretical predictions and afford redox potential shifts of -160 mV (heme IV), -90 mV (heme III), -70 mV (heme II), and +20 mV (heme I) relative to the experimental redox potentials for Cyt-c(3) in solution. SERR spectroscopic experiments reveal electric-field-induced changes of the redox potentials also for the structurally very similar Cyt-c(3) from Desulfovibrio vulgaris, although the shifts are somewhat smaller compared to Cyt-c(3) from D. gigas. This study suggests that electric-field-induced redox potential shifts may also occur upon binding to biomembranes or partner proteins and thus may affect biological electron transfer processes.

Biophysical Phenomena↗