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 ∼0.05 Å. When a symmetric back-to-back Morse potential is used to describe the tunneling particle vibrations in the reactant and product states, the D–A distribution width can be expanded up to ∼0.09 Å before the rates calculated using the linear approximation exceed the exact result by an order of magnitude. Incorporation of a more realistic anharmonic D–A potential, based on quantum calculations, includes the important electronic D–A repulsion energy so that the sampling of short D–A distances is reduced. This approach improves the linear approximation as long as the D–A distribution has a width less than ∼0.1 Å. The conditions for the validity of the linear approximation are found to be more fragile when the calculated kinetic isotope effect (KIE) and its temperature dependence are also taken into account

Investigations of the Low Frequency Modes of Ferric Cytochrome <i>c</i> Using Vibrational Coherence Spectroscopy

Femtosecond vibrational coherence spectroscopy is used to investigate the low frequency vibrational dynamics of the electron transfer heme protein, cytochrome <i>c</i> (cyt <i>c</i>). The vibrational coherence spectra of ferric cyt <i>c</i> 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. This assignment is based partially on the presence of a large heme ruffling distortion in the normal coordinate structural decomposition (NSD) analysis of the X-ray crystal structures. When the excitation wavelength is moved into the ∼421–435 nm region, the transient absorption increases along with the relative intensity of two modes near ∼55 and 30 cm^–1. The intensity of the mode near 44 cm^–1 appears to minimize in this region and then recover (but with an opposite phase compared to the blue excitation) when the laser is tuned to 443 nm. These observations are consistent with the superposition of both ground and excited state coherence in the 421–435 nm region due to the excitation of a weak porphyrin-to-iron charge transfer (CT) state, which has a lifetime long enough to observe vibrational coherence. The mode near 55 cm^–1 is suggested to arise from ruffling in a transient CT state that has a less ruffled heme due to its iron d⁶ configuration

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. <i>Biophys. J</i>. <b>2004</b>, <i>87</i>, 1881–1891) and optical (Gruia et al. <i>Biophys. J</i>. <b>2008</b>, <i>94</i>, 2252–2268) studies that had previously suggested the Fe–CN bond was photostable in MbCN. The photolysis of ferric MbCN takes place with a quantum yield of ∼75%, and the resonance Raman spectrum of the photoproduct observed in steady-state experiments as a function of laser power and sample spinning rate is identical to that of ferric Mb (metMb). The data are quantitatively analyzed using a simple model where cyanide is photodissociated and, although geminate rebinding with a rate of <i>k</i>_BA ≈ (3.6 ps)⁻¹ is the dominant process, some CN^– exits from the distal heme pocket and is replaced by water. Using independently determined values for the CN^– association rate, we find that the CN^– escape rate from the ferric myoglobin pocket to the solution at 293 K is <i>k</i>_out ≈ (1–2) × 10⁷ s^–1. This value is very similar to, but slightly larger than, the histidine gated escape rate of CO from Mb (1.1 × 10⁷ s^–1) under the same conditions. The analysis leads to an escape probability <i>k</i>_out/(<i>k</i>_out + <i>k</i>_BA) ∼ 10^–4, which is unobservable in most time domain kinetic measurements. However, the photolysis is surprisingly easy to detect in Mb using cw resonance Raman measurements. This is due to the anomalously slow CN^– bimolecular association rate (170 M^–1 s^–1), which arises from the need for water to exchange at the ferric heme binding site of Mb. In contrast, ferric HRP does not have a heme bound water molecule and its CN^– bimolecular association rate is larger by ∼10³, making the CN^– photolysis more difficult to observe

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. Thus, we have used quantum chemical calculations to account for the D–A interaction and developed an improved model for the analysis of experimental tunneling kinetics. Strong internal electric fields are also considered and found to contribute significantly to the compressive forces when the D–A distance distribution is positioned below the van der Waals contact distance. This model is applied to recent experiments on the wild type (WT) and a double mutant (DM) of soybean lipoxygenase-1 (SLO). The compressive force necessary to prepare the tunneling-active distribution in WT SLO is found to fall in the ∼ nN range, which greatly exceeds the measured values of molecular motor and protein unfolding forces. This indicates that ∼60–100 MV/cm electric fields, aligned along the D–A bond axis, must be generated by an enzyme conformational interconversion that facilitates the PCET tunneling reaction. Based on the absolute value of the measured tunneling rate, and using previously calculated values of the electronic matrix element, the population of this tunneling-active conformation is found to lie in the range 10^–5–10^–7, indicating this is a rare structural fluctuation that falls well below the detection threshold of recent ENDOR experiments. Additional analysis of the DM tunneling kinetics leads to a proposal that a disordered (high entropy) conformation could be tunneling-active due to its broad range of sampled D–A distances

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 <i>Tt</i> 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%). Geminate recombination of O₂ and NO in both of these proteins is very fast (∼1.4 × 10¹¹ s^–1) and highly efficient. We show that the distal heme pocket of the H-NOX system forms an efficient trap that limits the O₂ off-rate and determines the overall affinity. The distal pocket hydrogen bond, which appears to be stronger in the P115A mutant, may help retard the O₂ ligand from escaping into the solvent following either photoinduced or thermal dissociation. This, along with a strengthening of the Fe–O₂ bond that is correlated with the significant heme ruffing and saddling distortions, explains the unusually high O₂ affinity of WT <i>Tt</i> H-NOX and the even higher affinity found in the P115A mutant

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). Furthermore, through noncovalent tailoring of the chemistry of these quantum dots, we obtain water-stable quantum dots. For example, quantum dots with lysine groups bind strongly to DNA in solution and inhibit polymerase-based DNA strand synthesis. Finally, by virtue of their mesoscopic size, the quantum dots exhibit good cell permeability into living epithelial cells, but they do not enter the cell nucleus