Electrical unfolding of cytochrome during translocation through a nanopore constriction.

Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both.

Oxygen-Induced In Situ Manipulation of the Interlayer Coupling and Exciton Recombination in BiSe/MoS 2D Heterostructures.

Two-dimensional (2D) heterostructures are more than a sum of the parent 2D materials, but are also a product of the interlayer coupling, which can induce new properties. In this paper, we present a method to tune the interlayer coupling in BiSe/MoS 2D heterostructures by regulating the oxygen presence in the atmosphere, while applying laser or thermal energy. Our data suggest that the interlayer coupling is tuned through the diffusive intercalation and deintercalation of oxygen molecules.

Adiabatic Ligand Binding in Heme Proteins: Ultrafast Kinetics of Methionine Rebinding in Ferrous Cytochrome c.

The dynamics of methionine geminate recombination following photodissociation in ferrous cytochrome c is investigated over a broad temperature range. The kinetic response, above the solvent glass transition ( T), is nearly monoexponential and displays a weak temperature dependence. Below T, the rebinding kinetics are nonexponential and can be explained using a quenched distribution of enthalpic rebinding barriers, arising from a relatively narrow distribution of heme out-of-plane displacements.

Ultrafast CO Kinetics in Heme Proteins: Adiabatic Ligand Binding and Heavy Atom Tunneling.

The ultrafast kinetics of CO rebinding to carbon monoxide oxidation activator protein (ChCooA) are measured over a wide temperature range and compared with the kinetics of CO binding in other heme systems such as myoglobin (Mb) and hemoglobin (Hb). The Arrhenius prefactor for CO binding to ChCooA and protoheme (∼10 s) is similar to what is found for spin-allowed NO binding to heme proteins and is several orders of magnitude larger than the prefactor of Mb and Hb (∼10 s). This indicates that the CO binding reaction is adiabatic, in contrast to the commonly held view that it is nonadiabatic due to spin-forbidden (ΔS = 2) selection rules.

Tunneling Kinetics and Nonadiabatic Proton-Coupled Electron Transfer in Proteins: The Effect of Electric Fields and Anharmonic Donor-Acceptor Interactions.

A proper description of proton donor-acceptor (D-A) distance fluctuations is crucial for understanding tunneling in proton-coupled electron transport (PCET). The typical harmonic approximation for the D-A potential results in a Gaussian probability distribution, which does not appropriately reflect the electronic repulsion forces that increase the energetic cost of sampling shorter D-A distances. Because these shorter distances are the primary channel for thermally activated tunneling, the analysis of tunneling kinetics depends sensitively on the inherently anharmonic nature of the D-A interaction.

Peptide-Decorated Tunable-Fluorescence Graphene Quantum Dots.

We report here the synthesis of graphene quantum dots with tunable size, surface chemistry, and fluorescence properties. In the size regime 15-35 nm, these quantum dots maintain strong visible light fluorescence (mean quantum yield of 0.64) and a high two-photon absorption (TPA) cross section (6500 Göppert-Mayer units).

Proton-Coupled Electron Transfer and the "Linear Approximation" for Coupling to the Donor-Acceptor Distance Fluctuations.

The often-used "linear approximation" for treating the coupling of proton donor-acceptor (D-A) distance fluctuations to proton-coupled electron transfer tunneling reactions is systematically examined. The accuracy of this approximation is found to depend on the potential energy surfaces that are used to describe both the tunneling particle vibrations and the D-A coordinate probability distribution. Harmonic treatment of both the tunneling particle and the D-A coordinates results in a significant breakdown of the linear approximation when the width of the D-A distribution exceeds ∼0.

Wide-dynamic-range kinetic investigations of deep proton tunnelling in proteins.

Directional proton transport along 'wires' that feed biochemical reactions in proteins is poorly understood. Amino-acid residues with high pKa are seldom considered as active transport elements in such wires because of their large classical barrier for proton dissociation. Here, we use the light-triggered proton wire of the green fluorescent protein to study its ground-electronic-state proton-transport kinetics, revealing a large temperature-dependent kinetic isotope effect.

Kinetic Control of O2 Reactivity in H-NOX Domains.

Transient absorption, resonance Raman, and vibrational coherence spectroscopies are used to investigate the mechanisms of NO and O2 binding to WT Tt H-NOX and its P115A mutant. Vibrational coherence spectra of the oxy complexes provide clear evidence for the enhancement of an iron-histidine mode near 217 cm(-1) following photoexcitation, which indicates that O2 can be dissociated in these proteins. However, the quantum yield of O2 photolysis is low, particularly in the wild type (≲3%).

Deep proton tunneling in the electronically adiabatic and non-adiabatic limits: comparison of the quantum and classical treatment of donor-acceptor motion in a protein environment.

Analytical models describing the temperature dependence of the deep tunneling rate, useful for proton, hydrogen, or hydride transfer in proteins, are developed and compared. Electronically adiabatic and non-adiabatic expressions are presented where the donor-acceptor (D-A) motion is treated either as a quantized vibration or as a classical "gating" distribution. We stress the importance of fitting experimental data on an absolute scale in the electronically adiabatic limit, which normally applies to these reactions, and find that vibrationally enhanced deep tunneling takes place on sub-ns timescales at room temperature for typical H-bonding distances.

Investigations of the low frequency modes of ferric cytochrome c using vibrational coherence spectroscopy.

Femtosecond vibrational coherence spectroscopy is used to investigate the low frequency vibrational dynamics of the electron transfer heme protein, cytochrome c (cyt c). The vibrational coherence spectra of ferric cyt c have been measured as a function of excitation wavelength within the Soret band. Vibrational coherence spectra obtained with excitation between 412 and 421 nm display a strong mode at ~44 cm(-1) that has been assigned to have a significant contribution from heme ruffling motion in the electronic ground state.

Investigations of heme distortion, low-frequency vibrational excitations, and electron transfer in cytochrome c.

Cytochrome (cyt) c is an important electron transfer protein. The ruffling deformation of its heme cofactor has been suggested to relate to its electron transfer rate. However, there is no direct experimental evidence demonstrating this correlation.

Investigations of heme ligation and ligand switching in cytochromes p450 and p420.

It is generally accepted that the inactive P420 form of cytochrome P450 (CYP) involves the protonation of the native cysteine thiolate to form a neutral thiol heme ligand. On the other hand, it has also been suggested that recruitment of a histidine to replace the native cysteine thiolate ligand might underlie the P450 → P420 transition. Here, we discuss resonance Raman investigations of the H93G myoglobin (Mb) mutant in the presence of tetrahydrothiophene (THT) or cyclopentathiol (CPSH), and on pressure-induced cytochrome P420cam (CYP101), that show a histidine becomes the heme ligand upon CO binding.

Investigations of ferric heme cyanide photodissociation in myoglobin and horseradish peroxidase.

The photodissociation of cyanide from ferric myoglobin (MbCN) and horseradish peroxidase (HRPCN) has definitively been observed. This has implications for the interpretation of ultrafast IR (Helbing et al. Biophys.

Effect of DNA binding on geminate CO recombination kinetics in CO-sensing transcription factor CooA.

Carbon monoxide oxidation activator (CooA) proteins are heme-based CO-sensing transcription factors. Here we study the ultrafast dynamics of geminate CO rebinding in two CooA homologues, Rhodospirillum rubrum (RrCooA) and Carboxydothermus hydrogenoformans (ChCooA). The effects of DNA binding and the truncation of the DNA-binding domain on the CO geminate recombination kinetics were specifically investigated.

Vibrational coherence spectroscopy of the heme domain in the CO-sensing transcriptional activator CooA.

Femtosecond vibrational coherence spectroscopy was used to investigate the low-frequency vibrational dynamics of the heme in the carbon monoxide oxidation activator protein (CooA) from the thermophilic anaerobic bacterium Carboxydothermus hydrogenoformans (Ch-CooA). Low frequency vibrational modes are important because they are excited by the ambient thermal bath (k(B)T = 200 cm(-1)) and participate in thermally activated barrier crossing events. However, such modes are nearly impossible to detect in the aqueous phase using traditional spectroscopic methods.

Investigations of low-frequency vibrational dynamics and ligand binding kinetics of cystathionine beta-synthase.

Vibrational coherence spectroscopy is used to study the low frequency dynamics of the truncated dimer of human cystathionine beta-synthase (CBS). CBS is a pyridoxal-5'-phosphate-dependent heme enzyme with cysteine and histidine axial ligands that catalyzes the condensation of serine and homocysteine to form cystathionine. A strong correlation between the "detuned" coherence spectrum (which probes higher frequencies) and the Raman spectrum is demonstrated, and a rich pattern of modes below 200 cm(-1) is revealed.

Ultrafast dynamics of diatomic ligand binding to nitrophorin 4.

Nitrophorin 4 (NP4) is a heme protein that stores and delivers nitric oxide (NO) through pH-sensitive conformational change. This protein uses the ferric state of a highly ruffled heme to bind NO tightly at low pH and release it at high pH. In this work, the rebinding kinetics of NO and CO to NP4 are investigated as a function of iron oxidation state and the acidity of the environment.

Measurements of heme relaxation and ligand recombination in strong magnetic fields.

Heme cooling signals and diatomic ligand recombination kinetics are measured in strong magnetic fields (up to 10 T). We examined diatomic ligand recombination to heme model compounds (NO and CO), myoglobin (NO and O(2)), and horseradish peroxidase (NO). No magnetic field induced rate changes in any of the samples were observed within the experimental detection limit.

Low-frequency mode activity of heme: femtosecond coherence spectroscopy of iron porphine halides and nitrophorin.

The low-frequency mode activity of metalloporphyrins has been studied for iron porphine-halides (Fe(P)(X), X = Cl, Br) and nitrophorin 4 (NP4) using femtosecond coherence spectroscopy (FCS) in combination with polarized resonance Raman spectroscopy and density functional theory (DFT). It is confirmed that the mode symmetry selection rules for FCS are the same as for Raman scattering and that both Franck-Condon and Jahn-Teller mode activities are observed for Fe(P)(X) under Soret resonance conditions. The DFT-calculated low-frequency (20-400 cm (-1)) modes, and their frequency shifts upon halide substitution, are in good agreement with experimental Raman and coherence data, so that mode assignments can be made.

Temperature-dependent heme kinetics with nonexponential binding and barrier relaxation in the absence of protein conformational substates.

We present temperature-dependent kinetic measurements of ultrafast diatomic ligand binding to the "bare" protoheme (L(1)-FePPIX-L(2), where L(1) = H(2)O or 2-methyl imidazole and L(2) = CO or NO). We found that the binding of CO is temperature-dependent and nonexponential over many decades in time, whereas the binding of NO is exponential and temperature-independent. The nonexponential nature of CO binding to protoheme, as well as its relaxation above the solvent glass transition, mimics the kinetics of CO binding to myoglobin (Mb) but on faster time scales.

Analytical model of the optical response of periodically structured metallic films: Reply.

Some remarks are made on the validity of a commonly used analytical model based on the rigorous coupled wave analysis to describe the optical response of one-dimensional metallic gratings.