Grid-Based Projector Augmented Wave (GPAW) Implementation of Quantum Mechanics/Molecular Mechanics (QM/MM) Electrostatic Embedding and Application to a Solvated Diplatinum Complex.

A multiscale density functional theory-quantum mechanics/molecular mechanics (DFT-QM/MM) scheme is presented, based on an efficient electrostatic coupling between the electronic density obtained from a grid-based projector augmented wave (GPAW) implementation of density functional theory and a classical potential energy function. The scheme is implemented in a general fashion and can be used with various choices for the descriptions of the QM or MM regions. Tests on H2O clusters, ranging from dimer to decamer show that no systematic energy errors are introduced by the coupling that exceeds the differences in the QM and MM descriptions. Over 1 ns of liquid water, Born-Oppenheimer QM/MM molecular dynamics (MD) are sampled combining 10 parallel simulations, showing consistent liquid water structure over the QM/MM border. The method is applied in extensive parallel MD simulations of an aqueous solution of the diplatinum [Pt2(P2O5H2)4]4- complex (PtPOP), spanning a total time period of roughly half a nanosecond. An average Pt-Pt distance deviating only 0.01 Å from experimental results, and a ground-state Pt-Pt oscillation frequency deviating by <2% from experimental results were obtained. The simulations highlight a remarkable harmonicity of the Pt-Pt oscillation, while also showing clear signs of Pt-H hydrogen bonding and directional coordination of water molecules along the Pt-Pt axis of the complex.

Ab initio calculations of the electronic properties of polypyridine transition metal complexes and their adsorption on metal surfaces in the presence of solvent and counterions.

Os(II)/(III) and Co(II)/(III) polypyridine complexes in aqueous solution are robust molecular entities both in freely solute state and adsorbed on Au(111)- and Pt(111)-electrode surfaces. This class of robust coordination chemical compounds have recently been characterized by electrochemical scanning tunneling microscopy (in situ STM). The Os-complexes were found to display strong tunneling spectroscopic (STS) features at the level of resolution of the single molecule while STS features of the Co complexes, although clear, were much weaker. The data was framed by concise but phenomenological theory of interfacial electrochemical electron transfer extended to the electrochemical in situ STM configuration. With a view on first-principle insight into the in situ STM behavior of robust redox (as opposed to nonredox) molecules, we present in this report a density functional theory (DFT) study of the complexes in both free and adsorbate state, in either state exposed to both stoichiometric counterions and a large assembly of solvent water molecules. The oxidation states of the complexes were controlled, first by introducing chlorine counter atoms followed by spontaneous attraction of electrons from the complexes, also at first in electrostatically neutral form. Second, the solvent is found to provide strong dielectric screening of this charge transfer process and to be crucial for achieving the full chemically meaningful charge separated ionic oxidation states. The molecular charge and structure of the complexes in the presence of the solvent, are conserved upon adsorption, whereas the structural features of the different oxidation states are completely lost upon adsorption under vacuum conditions. Detailed microscopic insight such as offered by the present study will be important in molecular-based approaches to "smart" redox molecules enclosed in in situ STM or other nanoscale and single-molecules scale configurations in condensed matter environments.

Intrinsic multistate switching of gold clusters through electrochemical gating.

The electrochemical behavior of small metal nanoparticles is governed by Coulomb-like charging and equally spaced charge-transfer transitions. Using electrochemical gating at constant bias voltage, we show, for the first time, that individual nanoparticles can be operated as multistate switches in condensed media at room temperature, displaying distinct peak features in the tunneling current. The tunneling conductance increases with particle charge, suggesting that solvent reorganization and dielectric saturation become increasingly important.

Single-molecule conductance of redox molecules in electrochemical scanning tunneling microscopy.

Experimental data and theoretical notions are presented for 6-[1'-(6-mercapto-hexyl)-[4,4']bipyridinium]-hexane-1-thiol iodide (6V6) "wired" between a gold electrode surface and tip in an in situ scanning tunneling microscopy configuration. The viologen group can be used to "gate" charge transport across the molecular bridge through control of the electrochemical potential and consequently the redox state of the viologen moiety. This gating is theoretically considered within the framework of superexchange and coherent two-step notions for charge transport. It is shown here that the absence of a maximum in the Itunneling versus electrode potential relationship can be fitted by a "soft" gating concept. This arises from large configurational fluctuations of the molecular bridge linked to the gold contacts by flexible chains. This view is incorporated in a formalism that is well-suited for data analysis and reproduces in all important respects the 6V6 data for physically sound values of the appropriate parameters. This study demonstrates that fluctuations of isolated configurationally "soft" molecules can dominate charge transport patterns and that theoretical frameworks for compact monolayers may not be directly applied under such circumstances.

Double-tunnel nanoscale switch with a redox mediator: operational principles and tunneling spectroscopy.

A description of the physical mechanism and operation of a novel nanometric electronic switch [D.I. Gittins et al., Nature 2000 408, 67] is presented. New options for controlling the properties of this device are suggested and analyzed.

Deuterium isotope effect on the intramolecular electron transfer in Pseudomonas aeruginosa azurin.

Intramolecular electron transfer in azurin in water and deuterium oxide has been studied over a broad temperature range. The kinetic deuterium isotope effect, k(H)/k(D), is smaller than unity (0.7 at 298 K), primarily caused by the different activation entropies in water (-56.5 J K(-1) mol(-1)) and in deuterium oxide (-35.7 J K(-1) mol(-1)). This difference suggests a role for distinct protein solvation in the two media, which is supported by the results of voltammetric measurements: the reduction potential (E(0')) of Cu(2+/+) at 298 K is 10 mV more positive in D(2)O than in H(2)O. The temperature dependence of E(0') is also different, yielding entropy changes of -57 J K(-1) mol(-1) in water and -84 J K(-1) mol(-1) in deuterium oxide. The driving force difference of 10 mV is in keeping with the kinetic isotope effect, but the contribution to DeltaS from the temperature dependence of E(0') is positive rather than negative. Isotope effects are, however, also inherent in the nuclear reorganization Gibbs free energy and in the tunneling factor for the electron transfer process. A slightly larger thermal protein expansion in H(2)O than in D(2)O (0.001 nm K(-1)) is sufficient both to account for the activation entropy difference and to compensate for the different temperature dependencies of E(0'). Thus, differences in driving force and thermal expansion appear as the most straightforward rationale for the observed isotope effect.

An approach to long-range electron transfer mechanisms in metalloproteins: in situ scanning tunneling microscopy with submolecular resolution.

In situ scanning tunneling microscopy (STM) of redox molecules, in aqueous solution, shows interesting analogies and differences compared with interfacial electrochemical electron transfer (ET) and ET in homogeneous solution. This is because the redox level represents a deep indentation in the tunnel barrier, with possible temporary electronic population. Particular perspectives are that both the bias voltage and the overvoltage relative to a reference electrode can be controlled, reflected in spectroscopic features when the potential variation brings the redox level to cross the Fermi levels of the substrate and tip. The blue copper protein azurin adsorbs on gold(111) via a surface disulfide group. Well resolved in situ STM images show arrays of molecules on the triangular gold(111) terraces. This points to the feasibility of in situ STM of redox metalloproteins directly in their natural aqueous medium. Each structure also shows a central brighter contrast in the constant current mode, indicative of 2- to 4-fold current enhancement compared with the peripheral parts. This supports the notion of tunneling via the redox level of the copper atom and of in situ STM as a new approach to long-range electron tunneling in metalloproteins.

Structure and dynamics of the metal site of cadmium-substituted carboxypeptidase A in solution and crystalline states and under steady-state peptide hydrolysis.

PAC spectra (perturbed angular correlation of gamma-rays) of cadmium-substituted carboxypeptidase A (CPD) show that the enzyme in solution imposes a flexible, pH- and chloride-dependent coordination structure on the metal site, in contrast to what is found in the crystalline state. A much more restricted coordination geometry occurs for the steady-state peptide intermediates of Bz-Gly-l-Phe and Bz-Gly-Gly-l-Phe in solution, suggesting that substrate binding locks the structure in a rigid conformation. The results further indicate that the peptide intermediate has a six-coordinated metal coordination geometry with an OH- ligand at the solvent site and a carbonyl oxygen at an additional ligand site. In marked contrast, conformational rigidity is not induced by the inhibitor/poor substrate Gly-L-Tyr nor by the products of high turnover substrates, Bz-Gly, Bz-Gly-Gly, and L-Phe. These results are consistent with an intact scissile peptide bond in the enzyme-substrate complex of Bz-Gly-L-Phe and Bz-Gly-Gly-L-Phe. A single nuclear quadrupole interaction (NQI) is observed for the crystalline state of the enzyme between pH 5.7 and pH 9.4. This NQI agrees with calculations based on the metal coordination geometry for cadmium in crystalline CPD derived from X-ray diffraction studies. A single broad distribution of NQIs is observed for CPD in sucrose solutions and 0.1 M NaCl at pH values below 6.5. This NQI (NQI-1') has parameters very close to those for the crystalline state. The enzyme metal site, characterized by this NQI, is converted into two new enzyme metal sites over the pH range of 6.5-8.3. The metal coordination sphere of one of these has a NQI (NQI-1) with parameters similar to those at lower pH values (NQI-1') while the other NQI (NQI-2) is characterized by markedly different NQI parameters. Angular overlap model (AOM) calculations indicate that the coordination sites giving NQI-1' and NQI-1 both have a metal-bound water molecule while the coordination site giving NQI-2 has a metal-bound hydroxide ion. PAC results at pH 8.3-10.5 indicate that in this pH range the two metal coordination geometries related to NQI-1 and NQI-2 occur in a pH independent ratio of 2:1, with the one with the water ligand being the most abundant species. The observed pH-independent equilibrium between the two different metal coordination geometries for cadmium can be explained by an equilibrium between tautomeric forms of a hydrogen bond between the Glu-270 carboxyl group and the metal-bound water (Glu-270 COO-...(HOH)M <==> Glu-270 COOH...(OH-)M) being slow on the time scale of a PAC experiment, i.e., slower than 0.5 micros. We finally suggest that NQI-1' observed at low pH reflects an enzyme species containing a metal-coordinated water molecule and the protonated carboxyl group of Glu-270.

Solution structure of reduced plastocyanin from the blue-green alga Anabaena variabilis.

The three-dimensional solution structure of plastocyanin from Anabaena variabilis (A.v.PCu) has been determined by nuclear magnetic resonance spectroscopy. Sixty structures were calculated by distance geometry from 1141 distance restraints and 46 dihedral angle restraints. The distance geometry structures were optimized by simulated annealing and restrained energy minimization. The average rms deviation from the mean structure for the 20 structures with the lowest total energy is 1.25 A for the backbone atoms and 1.75 A for all heavy atoms. Overall, the global tertiary fold of A.v.PCu resembles those of other plastocyanins which have been structurally characterized by X-ray diffraction and NMR methods. This holds even though A.v.PCu is longer than any other known plastocyanins, contains far less invariant amino acid residues, and has an overall charge that differs considerably from those of other plastocyanins (+1 vs -9 +/- 1 at pH > or = 7). The most striking feature of the A.v. PCu structure is the absence of the beta-turn, formed at the remote site by residues (58)-(61) in most higher plant plastocyanins. The displacement caused by the absence of this turn is compensated for by an extension of the small helix [from Ala53(51) to Ser60(58) in A.v.PCu] found in other plastocyanins. Moreover, the extra residues of A.v.PCu from Pro77 to Asp79 form an appended loop. These two features allow A.v.PCu to retain almost the same global fold as observed in other plastocyanins. From a comparison with the structures of other plastocyanins it is concluded that the lack of negatively charged residues at the remote site, rather than the specific structure of A.v.PCu, is the main reason for the failure of the remote site of this plastocyanin to function as a significant electron transfer site.

pH and ionic strength effects on electron transfer rate constants and reduction potentials of the bacterial di-heme protein Pseudomonas stutzeri cytochrome c4.

We have investigated the ionic strength (0.1-0.5 M NaCl) and pH dependence (4.0-7.5) of the electron transfer (ET) rate constants for oxidation and reduction of the bacterial di-heme protein cytochrome c4 (cyt c4; Pseudomonas stutzeri, ATCC No. 11607) by [Co(bipy)3]3+/2+ (bipy = 2,2'-bipyridine). The kinetics is bi- or tri-phasic, and a mechanism based on cooperative ET at both hemes, slow intramolecular ET and electrostatically dominated inter-heme interaction is presently best in line with all the available data. The ionic strength and pH dependence of the rate constants and reduction potentials is weak. The rate constants mostly decrease by 0-50% in the ionic strength range 0.1-0.5 M. The macroscopic potentials decrease by < 10 mV. Three of the microscopic potentials increase by 10-25 mV, while the fourth one decreases by 50 mV, but the accuracy of the microscopic reduction potential values is low. There is no pH dependence of the rate constants in the range 6.0-7.5, but most rate constants drop to half the 6.0-7.5 value in the range 4.0-6.0, leaving the reduction potentials almost unaffected. The small effects are unexpected in view of the highly charged and strongly dipolar character, and the many hydrogen bond contacts of cyt c4. These small effects must be related to the detailed rather the overall charge distribution of cyt c4.

Crystallization and preliminary crystallographic investigations of cytochrome c4 from Pseudomonas stutzeri.

Cytochrome c(4) from the bacterium Pseudomonas stutzeri has been crystallized and X-ray diffraction data have been collected to 2.2 A resolution. The crystals belong to the monoclinic system, space group P2(1) with cell parameters a = 49.49, b = 58.58, c = 63.51 A and beta = 96.96 degrees. The crystals contain two molecules it, the asymmetric unit. Cytochrome c(4) is known to contain two covalently bound haem groups per molecule and positions of the four haem Fe atoms in the asymmetric unit were determined from native anomalous-dispersion differences. The shortest Fe-Fe distances found in the crystal were 15.8, 16.9 and 19.0 A.

Electron transfer and spectral alpha-band properties of the di-heme protein cytochrome c4 from Pseudomonas stutzeri.

Cytochrome c4 is a 190-residue protein active in the aerobic and anaerobic respiration of several bacteria. We have isolated Pseudomonas stutzeri (ATCC no. 11607) cytochrome c4 by an optimized growth procedure following factorial design. The ultraviolet/visible spectra of reduced cytochrome c4 have a composite alpha/beta band which can be resolved into six components. One of these seems to be specific for the high-potential heme group. The kinetics for full oxidation and reduction with the two inorganic redox couples, [Co(terpy)2]2+/3+ and [Co(bipy)3]2+/3+, is formally compatible with either bi- or tri-exponential kinetics. The former would be in line with weak interaction between the heme groups, the latter with notable interaction effects. Arguments in favour of the latter and a cooperative two-electron transfer pattern are given. All phases are approximately proportional to the Co-complex concentration, implying that intramolecular electron transfer in this time range is unlikely. The rate constants are in the range (0.7-80) x 10(4) M-1 s-1 at pH = 7.6 (Tris) and 0.1 M NaCl and very little dependent on the ionic strength in the range 0.1-0.3 M. The reduction potentials could be calculated from the forward and reverse rate constant ratios. The values are 241 +/- 5 and 328 +/- 2 mV (Nernst hydrogen electrode) if bi-exponential kinetics is used and interaction between the heme groups disregarded. The intrinsic microscopic reduction potential values are closer when the tri-exponential, cooperative model is used as this model transfers 30-40 mV to electrostatically dominated interaction potentials. The overall electron transfer pattern can be related to the recently determined crystal structure of the P. stutzeri cytochrome c4.

Relationship of secretion pattern and MPB70 homology with osteoblast-specific factor 2 to osteitis following Mycobacterium bovis BCG vaccination.

Significant homology was found between MPB70 and each of four repeat domains of osteoblast-specific factor 2 (OSF-2). Two internal homology regions within each repeat domain of OSF-2 presumed to be related to the active site(s) of this bone adhesion molecule showed the highest homology. A literature search concerning osteitis after Mycobacterium bovis BCG vaccination in neonates revealed that MPB70-high-producer substrains were associated with an increased incidence of osteitis following vaccination. These observations indicate that the function of MPB70 is related to the interaction between bacilli and the host following vaccination or infection with mycobacteria.

Synthesis and characterization of Desulfovibrio gigas rubredoxin and rubredoxin fragments.

The 52-residue Desulfovibrio gigas rubredoxin peptide chain has been synthesized and a procedure for chain folding around iron(II) developed. The folded, stable synthetic rubredoxin can be subjected to purification, and reversibly oxidized and reduced. Ultraviolet/visible absorption and CD spectra of both forms show all the same features as native D. gigas rubredoxin, and the symmetric and asymmetric Fe-S stretching bands in the resonance Raman spectrum can be identified. In addition, the matrix-assisted laser desorption mass spectrum of a peptide sample exposed to trace amounts of iron is dominated by a peak at 5735Da very close to the value for the calculated molecular mass. Details in the ultraviolet/visible bandshape and mass spectrum, however, indicate remaining impurities. In comparison, a previously synthesized 25-residue rubredoxin fragment with the non-conserved positions 13-35 and 51-52 omitted and Val5-Glu50 anchored via glycine folds gives the correct molecular mass and ultraviolet/visible spectrum, but is much more labile than the 52-residue protein. This shows that non-conserved residues are crucial in protein folding and that chemical metalloprotein synthesis offers alternative prospects to microbiological protein engineering.

Evidence for absence of the MPB64 gene in some substrains of Mycobacterium bovis BCG.

Substrains of Mycobacterium bovis BCG have been divided in two major groups, high producers and low producers of the secreted proteins MPB64 and MPB70. Of these, Mycobacterium tuberculosis secretes only the analog MPT64 during growth on Sauton medium. It has been confirmed that high-producer and low-producer substrains of BCG as well as M. tuberculosis contain the gene for the MPB/MPT70 protein. By contrast, polymerase chain reaction and hybridization experiments are reported here which indicate that the MPB64 gene is absent in the BCG substrains Copenhagen, Pasteur, Glaxo, and Tice, in which previous methods did not permit distinction between secretion of small amounts or absence of the protein in culture fluids.

Redox potential and electrostatic effects in competitive inhibition of dual-path electron transfer reactions of spinach plastocyanin.

Redox inactive ions with high positive charges lower the rate constant for oxidation of several plant plastocyanins (PC) by small positively charged inorganic reaction partners. The rate constant decrease is commonly attributed to competitive inhibition where the redox inactive ions are bound to the negatively charged remote electron transfer (ET) site of PC and block this site sterically. We have investigated the effects of the inhibitor [NH3)5Co(NH2)Co(NH3)5]5+ on the ET reactions of spinach PC with [Co(phen)3]3+ (phen = 1,10-phenanthroline) and the electrically neutral analogue [Co(phen-SO3)3] (phen-SO3 = 5-sulfonato-1,10-phenanthroline) at the ionic strengths mu = 0.1 M and 0.03 M. Inhibition of the [Co(phen)3]3+ reactions is notably smaller for PC(II) reduction than for PC(I) oxidation. This is indicative of a redox potential increase of PC(II)/PC(I) on inhibitor attachment. The effect amounts to 16 mV at mu = 0.1 M and 31 mV at mu = 0.03 M. These data, and analysis in terms of ET theory show that inhibition cannot be caused solely by steric blocking. Driving force and interreactant electrostatic work terms are equally important. The PC(I)/[Co(phen-SO3)3] reaction exhibits a more entangled pattern. The rate constant first increases slightly with increasing inhibitor concentration, then drops, and approaches a constant value not far from the original value. This pattern is in line with association between the negatively charged -SO3- groups of the Co(III) complex and the inhibitor, and ET of the associate at both ET sites of PC.

Synthesis and characterization of a 25-residue rubredoxin(II)-like metalloprotein and its valine-leucine mutant.

An iron-sulfur metalloprotein containing the 5-12 and 35-50 residues of Desulfovibrio gigas rubredoxin has been synthesized by Fmoc solid phase peptide synthesis and subsequent peptide folding. A Gly links the two residue chains between Val-5 and Glu-50. Sybyl Tripos structure optimization indicates only minor structural changes of the folded synthetic protein compared to the similar residue positions in the native protein. The UV-VIS spectrum of the reduced synthetic protein is very similar to that of native D. gigas rubredoxin and the molecular mass determined by laser mass spectrometry has the expected value (+/- 2D). No metal is transferred to the gas phase by the laser beam merely by mixing the peptide and iron(II), substantiating that the folding procedure is a necessary pre-requisite for protein formation. The Val-->Leu41 chemical mutant has also been synthesized and behaves in a closely similar fashion.

Inhomogeneous broadening and kinetic carbon monoxide isotope effects in low-temperature carbon monoxide recombination with myoglobin and hemoglobin.

We have analyzed the non-exponential kinetics, the temperature variation, and the CO isotope effects of the CO recombination reactions with myoglobin and single-chain hemoglobin. The analysis rests on multiphonon quantum-mechanical chemical-rate theory combined with static inhomogeneous broadening of either the reorganization free energy or the reaction Gibbs free energy. The simplest specific model which can account for all the data contains an inhomogeneous distribution function of width 0.2-0.3 eV, independent of temperature down to the tunnel transition at about 20 K, two discrete nuclear coordinates of low vibrational frequency (60-150 cm-1) representing iron-heme and CO bending motion, the CO stretching motion of frequency about 2000 cm-1, and additional inhomogeneous broadening of the protein and CO bending configuration below the tunnel transition temperature. The model appears somewhat involved but in return provides corresponding insight in the dynamics of this important class of processes.

Resonance effects in strongly exothermic long-range electron transfer and their possible implications for the behaviour of site-directed mutant proteins.

Long-range electron transfer investigations of hemoproteins, blue copper and iron-sulphur proteins frequently rest on electronically excited metal centres. When the excitation energy approaches the oxidation or reduction potentials of intermediate residues the superexchange view normally used, however, fails and a variety of new dynamic features arise. These all involve population of the intermediate cation or anion residue states which can be partially or wholly vibrationally relaxed. We discuss suitable views and a new theoretical formalism for these phenomena. We also note some important implications for site-directed mutagenesis in long-range, strongly exothermic electron transfer processes.

Effects of NO2-modification of Tyr83 on the reactivity of spinach plastocyanin with cytochrome f.

We have investigated the electron transfer (ET) reactions between turnip cytochrome f, and the native and NO2-Tyr83-modified forms of spinach plastocyanin (PCu) at 10.0 degrees C and ionic strength 0.200 M(NaCl), in both directions as a function of pH. The PCu(II)/cytochrome f(II) rate constants in the pH-range 4-6.8 reflect active and remote binding site protonation. At higher pH, NO2-Tyr83 and positively charged residues on cytochrome f are deprotonated, and both native and NO2-modified PCu exhibit a composite rate constant variation in this pH range. When framed by ET theory this pattern is fully understandable in terms of variations in reduction potentials and electrostatic interactions, caused by the protonation equilibria. The rate constant ratio knitro/knative is, however, only 1.04 for the PCu(II)/cytochrome f(II) reactions in spite of a 18 mV higher reduction potential for NO2-Tyr83-modified PCu. This is much lower than the value of 1.42 expected from ET theory solely on the basis of such a reduction potential effect. A similar effect is seen for PCu(I)/cytochrome f(III) for which the low-pH knitro/knative ratio is 0.51. Notable but smaller effects are also observed for the small reaction partners [Fe(CN)6]3-/4- and [Co(phen)3]3+/2+. The effect of NO2-modification in addition to the reduction potential effect can be resolved into a small reorganization energy increase around the copper atom and a smaller electronic transmission coefficient for ET through the Cu/Cys84/Tyr83 sequence. The former effect dominates in the reactions with the small reaction partners, while the electronic effects contribute significantly for PCu/cytochrome f, supporting the concept that the PCu/cytochrome f ET is at the remote PCu binding site.