Electron tunneling through organic molecules in frozen glasses.

Reaction rates extracted from measurements of donor luminescence quenching by randomly dispersed electron acceptors reveal an exponential decay constant of 1.23 per angstrom for electron tunneling through a frozen toluene glass (with a barrier to tunneling of 1.4 electron volts).

Many faces of the unfolded state: conformational heterogeneity in denatured yeast cytochrome C.

We have measured fluorescence energy-transfer (FET) kinetics from a dansyl fluorophore (Dns) introduced by derivatization of a Cys side-chain to the Fe(III) heme covalently attached to unfolded yeast iso-1 cytochrome c (cyt). To gain a global picture of the unfolded state, we examined variants with the fluorophore attached on three different helices (K4C, E66C, K99C) and in three different loops (H39C, D50C, L85C). Analysis of the FET kinetics data gave distributions of distances between the fluorescent donor and acceptor; these distributions demonstrate that the guanidine hydrochloride (GuHCl)-denatured polypeptide ensemble is not a simple random coil.

Zinc-porphyrin Solvation in folded and unfolded states of Zn-cytochrome c.

After a brief review of the use of photochemical triggers and heme metal substitution to probe the folding dynamics of cytochrome c, we present new results on the photophysics and photochemistry of folded and unfolded states of the zinc-substituted protein (Zn-cyt c). Our measurements of Zn-cyt c triplet state decay kinetics reveal a systematic isotope effect on lifetimes: the decay in the folded protein (tau(H)2(O) approximately 10 ms) is only modestly affected by isotopically substituted buffers (k(H)2(O)/k(D)2(O) = 1.2), whereas a reduced triplet lifetime (approximately 1.

Inner-sphere electron-transfer reorganization energies of zinc porphyrins.

Inner-sphere electron-transfer reorganization energies of Zn(protoporphyrin IX) and Zn(octaethylporphyrin) are determined from band-shape analyses of the first ionization obtained by gas-phase valence photoelectron spectroscopy. The experimentally determined total inner-sphere reorganization energies for self-exchange (120-140 meV) indicate that structural changes upon oxidation are largely confined to the porphyrin ring, and substituents on the ring or solvent and other environmental factors make smaller contributions. Computational estimates by different models vary over a wide range and are sensitive to numerical precision factors for these low reorganization energies.

Alpha-synuclein structures from fluorescence energy-transfer kinetics: implications for the role of the protein in Parkinson's disease.

Parkinson's disease is associated with the deposition and accumulation of alpha-synuclein fibrils in the brain. A30P and A53T mutations have been linked to the early-onset familial disease state. Time-resolved tryptophan fluorescence energy-transfer measurements have been used to probe the structures of pseudo-wild-type and mutant (A30P) alpha-synucleins at physiological pH (7.

Conformational states of cytochrome P450cam revealed by trapping of synthetic molecular wires.

Members of the ubiquitous cytochrome P450 family catalyze a vast range of biologically significant reactions in mammals, plants, fungi, and bacteria. Some P450s display a remarkable promiscuity in substrate recognition, while others are very specific with respect to substrate binding or regio and stereo-selective catalysis. Recent results have suggested that conformational flexibility in the substrate access channel of many P450s may play an important role in controlling these effects.

Mimicking protein-protein electron transfer: voltammetry of Pseudomonas aeruginosa azurin and the Thermus thermophilus Cu(A) domain at omega-derivatized self-assembled-monolayer gold electrodes.

Well-defined voltammetric responses of redox proteins with acidic-to-neutral pI values have been obtained on pure alkanethiol as well as on mixed self-assembled-monolayer (SAM) omega-derivatized alkanethiol/gold bead electrodes. Both azurin (P. aeruginosa) (pI = 5.

Alpha-synuclein: stable compact and extended monomeric structures and pH dependence of dimer formation.

The protein alpha-synuclein, implicated in Parkinson's disease, was studied by combining nano-electrospray ionization (N-ESI) mass spectrometry and ion mobility. It was found that both the charge-state distribution in the mass spectra and the average protein shape deduced from ion mobility data, depend on the pH of the spray solution. Negative-ion N-ESI of pH 7 solutions yielded a broad charge-state distribution from -6 to -16, centered at -11, and ion mobility data consistent with extended protein structures.

Cobaltocene-mediated catalytic monooxygenation using holo and heme domain cytochrome P450 BM3.

The feasibility of replacing NADPH with 1,1'-dicarboxycobaltocene in the catalytic cycle of cytochrome P450 BM3 has been explored. Using the holoprotein, the surrogate mediator was observed to reduce both the FAD and FMN in the reductase domain, as well as the iron in the heme domain. In an electrochemical system, the mediator was able to support lauric acid hydroxylation at a rate of 16.

Reduction of dioxygen catalyzed by pyrene-wired heme domain cytochrome P450 BM3 electrodes.

We have electronically wired the cytochrome P450 BM3 heme domain to a graphite electrode with the use of a pyrene-terminated tether. AFM images clearly reveal that pyrene-wired enzyme molecules are adsorbed to the electrode surface. The enzyme-electrode system undergoes rapid and reversible electron transfer, displaying a standard rate constant higher than that of any other P450-electrode system.

Excited-state dynamics of fac-[ReI(L)(CO)3(phen)]+ and fac-[ReI(L)(CO)3(5-NO2-phen)]+ (L = imidazole, 4-ethylpyridine; phen = 1,10-phenanthroline) complexes.

The nature and dynamics of the lowest excited states of fac-[Re(I)(L)(CO)(3)(phen)](+) and fac-[Re(I)(L)(CO)(3)(5-NO(2)-phen)](+) [L = Cl(-), 4-ethyl-pyridine (4-Etpy), imidazole (imH); phen = 1,10-phenanthroline] have been investigated by picosecond visible and IR transient absorption spectroscopy in aqueous (L = imH), acetonitrile (L = 4-Etpy, imH), and MeOH (L = imH) solutions. The phen complexes have long-lived Re(I) --> phen (3)MLCT excited states, characterized by CO stretching frequencies that are upshifted relative to their ground-state values and by widely split IR bands due to the out-of-phase A'(2) and A"nu(CO) vibrations. The lowest excited states of the 5-NO(2)-phen complexes also have (3)MLCT character; the larger upward nu(CO) shifts accord with much more extensive charge transfer from the Re(I)(CO)(3) unit to 5-NO(2)-phen in these states.

Amphiphilic corroles bind tightly to human serum albumin.

Amphiphilic 2,17-bis-sulfonato-5,10,15(trispentafluorophenyl)corrole (2) and its Ga and Mn complexes (2-Ga and 2-Mn) form tightly bound noncovalent conjugates with human serum albumin (HSA). Protein-induced changes in the electronic absorption, emission, and circular dichroism spectra of these corroles, as well as results obtained from HPLC profiles of the conjugates and selective fluorescence quenching of the single HSA tryptophan, are interpreted in terms of multiple corrole:HSA binding sites. High-affinity binding sites, close to the unique tryptophan, are fully occupied at very low concentrations.

Oxoiron(IV) in chloroperoxidase compound II is basic: implications for P450 chemistry.

With the use of x-ray absorption spectroscopy, we have found that the Fe-O bond in chloroperoxidase compound II (CPO-II) is much longer than expected for an oxoiron(IV) (ferryl) unit; notably, the experimentally determined bond length of 1.82(1) A accords closely with density functional calculations on a protonated ferryl (Fe(IV)-OH, 1.81 A).

Electron-transfer chemistry of Ru-linker-(heme)-modified myoglobin: rapid intraprotein reduction of a photogenerated porphyrin cation radical.

We report the synthesis and characterization of RuC7, a complex in which a heme is covalently attached to a [Ru(bpy)(3)](2+) complex through a -(CH(2))(7)- linker. Insertion of RuC7 into horse heart apomyoglobin gives RuC7Mb, a Ru(heme)-protein conjugate in which [Ru(bpy)(3)](2+) emission is highly quenched. The rate of photoinduced electron transfer (ET) from the resting (Ru(2+)/Fe(3+)) to the transient (Ru(3+)/Fe(2+)) state of RuC7Mb is >10(8) s(-1); the back ET rate (to regenerate Ru(2+)/Fe(3+)) is 1.

The stability of the cytochrome c scaffold as revealed by NMR spectroscopy.

NMR spectroscopy was used to study the effect of guanidinium chloride on the unfolding of horse heart and yeast iso-1 cytochrome c under mild alkaline conditions. The structural changes on the horse heart protein were detected through NOESY (Nuclear Overhauser Effect SpectroscopY) experiments whereas (15)N-(1)H heteronuclear NMR was used to monitor the behavior of the yeast protein. The latter represents the first characterization through (15)N-(1)H heteronuclear NMR spectroscopy of the guanidinium chloride induced unfolding of mitochondrial cytochrome c.

X-ray magnetic circular dichroism of Pseudomonas aeruginosa nickel(II) azurin.

We show that X-ray magnetic circular dichroism (XMCD) can be employed to probe the oxidation states and other electronic structural features of nickel active sites in proteins. As a calibration standard, we have measured XMCD and X-ray absorption (XAS) spectra for the nickel(II) derivative of Pseudomonas aeruginosa azurin (NiAz). Our analysis of these spectra confirms that the electronic ground state of NiAz is high-spin (S = 1); we also find that the L(3)-centroid energy is 853.

Electron tunneling in rhenium-modified Pseudomonas aeruginosa azurins.

Laser flash-quench methods have been used to generate tyrosine and tryptophan radicals in structurally characterized rhenium-modified Pseudomonas aeruginosa azurins. Cu(I) to "Re(II)" electron tunneling in Re(H107) azurin occurs in the microsecond range. This reaction is much faster than that studied previously for Cu(I) to Ru(III) tunneling in Ru(H107) azurin, suggesting that a multistep ("hopping") mechanism might be involved.

Electron tunneling through proteins.

Electron transfer processes are vital elements of energy transduction pathways in living cells. More than a half century of research has produced a remarkably detailed understanding of the factors that regulate these 'currents of life'. We review investigations of Ru-modified proteins that have delineated the distance- and driving-force dependences of intra-protein electron-transfer rates.

Photoswitchable luminescence of rhenium(i) tricarbonyl diimines.

The synthesis, characterization, and X-ray crystal structures of [Re(diimine)(CO)(3)(dpe)](PF(6)) (dpe = 1,2-di(4-pyridyl)ethylene) compounds are reported. The cis-dpe complexes exhibit yellow luminescence after UV excitation, whereas the trans-dpe counterparts are nonluminescent. The luminescence quantum yields of the cis-dpe complexes are strongly dependent on the identity of the diimine ligand.

NMR-validated structural model for oxidized Rhodopseudomonas palustris cytochrome c(556).

The structure of oxidized Rhodopseudomonas palustris cytochrome c(556) has been modeled after that of high-spin cytochrome c' from the same bacterium, the latter being the protein with the greatest sequence identity (35%) among all sequenced proteins in the genomes. The two proteins differ in the number of ligands to iron and in spin state, the former being six-coordinate low-spin and the latter five-coordinate high-spin. In order to validate this modeled structure, several structural restraints were obtained by performing a restricted set of NMR experiments, without performing a complete assignment of the protein signals.

Spectroscopy and reactivity of a photogenerated tryptophan radical in a structurally defined protein environment.

Near-UV irradiation of structurally characterized [Re(I)(CO)3(1,10-phenanthroline)(Q107H)](W48F/Y72F/H83Q/Y108W)AzM(II) [Az = Pseudomonas aeruginosa azurin, M = Cu, Zn]/[Co(NH3)5Cl]Cl2 produces a tryptophan radical (W108*) with unprecedented kinetic stability. After rapid formation (k = 2.8 x 106 s-1), the radical persists for more than 5 h at room temperature in the folded ReAzM(II) structure.

Nanosecond photoreduction of cytochrome p450cam by channel-specific Ru-diimine electron tunneling wires.

We report the synthesis and characterization of Ru-diimine complexes designed to bind to cytochrome p450cam (CYP101). The sensitizer core has the structure [Ru(L(2))L'](2+), where L' is a perfluorinated biphenyl bridge (F(8)bp) connecting 4,4'-dimethylbipyridine to an enzyme substrate (adamantane, F(8)bp-Ad), a heme ligand (imidazole, F(8)bp-Im), or F (F(9)bp). The electron-transfer (ET) driving force (-deltaG degrees ) is varied by replacing the ancillary 2,2'-bipyridine ligands with 4,4',5,5'-tetramethylbipyridine (tmRu).

Gold electrodes wired for coupling with the deeply buried active site of Arthrobacter globiformis amine oxidase.

Diethylaniline-terminated oligo(phenyl-ethynyl)-thiol (DEA-OPE-SH) wires on Au-bead electrodes facilitate electron tunneling to and from the deeply buried topaquinone (TPQ) cofactor in Arthrobacter globiformis amine oxidase (AGAO). Reversible cyclic voltammograms were observed when AGAO was adsorbed onto this DEA-OPE-SAu surface: the 2e-/2H+ reduction potential is -140 mV versus SCE.

15N-1H Residual dipolar coupling analysis of native and alkaline-K79A Saccharomyces cerevisiae cytochrome c.

Residual dipolar couplings (RDCs) and pseudocontact shifts are experimentally accessible properties in nuclear magnetic resonance that are related to structural parameters and to the magnetic susceptibility anisotropy. We have determined RDCs due to field-induced orientation of oxidized-K79A and reduced cytochrome c at pH 7.0 and oxidized-K79A cytochrome c at pH 11.