Tyr25, Tyr58 and Trp133 of Escherichia coli bacterioferritin transfer electrons between iron in the central cavity and the ferroxidase centre

Ferritins are 24meric proteins that overcome problems of toxicity, insolubility and poor bioavailability of iron in all types of cells by storing it in the form of a ferric mineral within their central cavities. In the bacterioferritin (BFR) from Escherichia coli iron mineralization kinetics have been shown to be dependent on an intra-subunit catalytic diiron cofactor site (the ferroxidase centre), three closely located aromatic residues and an inner surface iron site. One of the aromatic residues, Tyr25, is the site of formation of a transient radical, but the roles of the other two residues, Tyr58 and Trp133, are unknown. Here we show that these residues are important for the rates of formation and decay of the Tyr25 radical and decay of a secondary radical observed during Tyr25 radical decay. The data support a mechanism in which these aromatic residues function in electron transfer from the inner surface site to the ferroxidase centre

Conformational changes in quadruplex oligonucleotide structures probed by Raman spectroscopy

Quadruplex structures are higher order structures formed by guanine-rich oligonucleotides. In the present study, temperature-induced conformational changes in the quadruplex structures of aptamers and other guanine-rich oligonucleotides are probed by Raman spectroscopy. In particular, dramatic changes in the fingerprint region are observed in the spectra of thrombin binding aptamer at higher temperatures. These changes are accompanied by a decrease in the intensity of the 1480 cm−1 peak (attributed to C8 = N7-H2), which is diagnostic of the quadruplex structure. We also show that these changes can be reversed (to a certain extent) by addition of K+ ions

Redox-Induced Conformational Switching in Photosystem-II-Inspired Biomimetic Peptides: A UV Resonance Raman Study

Long-distance electron transfer (ET) plays a critical role in solar energy conversion, DNA synthesis, and mitochondrial respiration. Tyrosine (Y) side chains can function as intermediates in these reactions. The oxidized form of tyrosine deprotonates to form a neutral tyrosyl radical, Y^•, a powerful oxidant. In photosystem II (PSII) and ribonucleotide reductase, redox-active tyrosines are involved in the proton-coupled electron transfer (PCET) reactions, which are key in catalysis. In these proteins, redox-linked structural dynamics may play a role in controlling the radical’s extraordinary oxidizing power. To define these dynamics in a structurally tractable system, we have constructed biomimetic peptide maquettes, which are inspired by PSII. UV resonance Raman studies were conducted of ET and PCET reactions in these β-hairpins, which contain a single tyrosine residue. At pH 11, UV photolysis induces ET from the deprotonated phenolate side chain to solvent. At pH 8.5, interstrand proton transfer to a π-stacked histidine accompanies the Y oxidation reaction. The UV resonance Raman difference spectrum, associated with Y oxidation, was obtained from the peptide maquettes in D₂O buffers. The difference spectra exhibited bands at 1441 and 1472 cm^–1, which are assigned to the amide II′ (CN) vibration of the β-hairpin. This amide II′ spectral change was attributed to substantial alterations in amide hydrogen bonding, which are coupled with the Y/Y^• redox reaction and are reversible. These experiments show that ET and PCET reactions can create new minima in the protein conformational landscape. This work suggests that charge-coupled conformational changes can occur in complex proteins that contain redox-active tyrosines. These redox-linked dynamics could play an important role in control of PCET in biological oxygen evolution, respiration, and DNA synthesis

Zinc-Substituted Cytochrome P450_cam: Characterization of Protein Conformers F420 and F450 by Photoinduced Electron Transfer

Metal substitution of heme proteins is widely applied in the study of biologically relevant electron transfer (ET) reactions. It has been shown that many modified proteins remain in their native conformation and can provide useful insights into the molecular mechanism of electron transfer between the native protein and its substrates. We investigated ET reactions between zinc-substituted cytochrome P450_cam and small organic compounds such as quinones and ferrocene, which are capable of accessing the protein’s hydrophobic channel and binding close to the active site, like its native substrate, camphor. Following the substitution method developed by Gunsalus and co-workers [Wagner, G. C., et al. (1981) <i>J. Biol. Chem. 256</i>, 6262–6265], we have identified two dominant forms of the zinc-substituted protein, F450 and F420, that exhibit different photophysical and photochemical properties. The ET behavior of F420 suggests that hydrophobic redox-active ligands are able to penetrate the hydrophobic channel and place themselves in the direct vicinity of the Zn-porphyrin. In contrast, the slower ET quenching rates observed in the case of F450 indicate that the association is weak and occurs outside of the protein channel. Therefore, we conclude that F420 corresponds to the open structure of the native cytochrome P450_cam while F450 has a closed or partially closed channel that is characteristic of the camphor-containing cytochrome P450_cam. The existence of two distinct conformers of Zn-bound P450_cam is consistent with the findings of Goodin and co-workers [Lee, Y.-T., et al. (2010) <i>Biochemistry 49</i>, 3412–3419] and has significant consequences for future electron transfer studies on this popular metalloenzyme

Vibrational State Dependence of Interfacial Electron Transfer: Hot Electron Injection from the S₁ State of Azulene into TiO₂ Nanoparticles

Strong dependence of the quantum yield of electron injection, Φ_injection, on the excess vibrational energy of the short-lived S₁ excited state of the donor was observed for a carboxyazulene chromophore, 6-MeAz-2-COOH, bound to the surface of colloidal TiO₂. The oxidation state of 6-MeAz-2-COOH was aligned with the CB of TiO₂ in such manner that the lowest vibrational levels of the S₁ state were below the band edge. Two distinct electron injection regimes were observed: a long wavelength one which was attributed to a low yield direct injection into trap sites and a short wavelength one corresponding to a much higher yield injection into the bulk CB of the TiO₂ nanoparticles. There is a 9-fold increase of Φ_injection as λ_excitation decreases from 690 to 525 nm, with the steepest, 4-fold rise occurring between 585 and 550 nm, that is, when the excess energy is approximately equivalent to 3 quanta of skeletal vibrations of the donor. The charge recombination kinetics in the 6-MeAz-2-COOH@TiO₂ system is also different for the low energy (λ > 585 nm) and high energy (λ < 585 nm) excitation of the donor. This behavior is consistent with excess energy dependent spatial range of injection and different trapping sites that are accessible to the “cold” and “hot” electrons, with the latter exhibiting a broader distribution of lifetimes and 24-times higher long-term yield at 30 ps after the excitation. Through demonstrating that it is possible to harvest electrons from unrelaxed, vibrationally hot donor states under ambient conditions in solution, these results open interesting directions for new developments in photovoltaics and photocatalysis

Redox-Dependent Structural Coupling between the α2 and β2 Subunits in <i>E. coli</i> Ribonucleotide Reductase

Ribonucleotide reductase (RNR) catalyzes the production of deoxyribonucleotides in all cells. In <i>E. coli</i> class Ia RNR, a transient α2β2 complex forms when a ribonucleotide substrate, such as CDP, binds to the α2 subunit. A tyrosyl radical (Y122O•)-diferric cofactor in β2 initiates substrate reduction in α2 via a long-distance, proton-coupled electron transfer (PCET) process. Here, we use reaction-induced FT-IR spectroscopy to describe the α2β2 structural landscapes, which are associated with dATP and hydroxyurea (HU) inhibition. Spectra were acquired after mixing <i>E. coli</i> α2 and β2 with a substrate, CDP, and the allosteric effector, ATP. Isotopic chimeras, ¹³Cα2β2 and α2¹³Cβ2, were used to define subunit-specific structural changes. Mixing of α2 and β2 under turnover conditions yielded amide I (CO) and II (CN/NH) bands, derived from each subunit. The addition of the inhibitor, dATP, resulted in a decreased contribution from amide I bands, attributable to β strands and disordered structures. Significantly, HU-mediated reduction of Y122O• was associated with structural changes in α2, as well as β2. To define the spectral contributions of Y122O•/Y122OH in the quaternary complex, ²H₄ labeling of β2 tyrosines and HU editing were performed. The bands of Y122O•, Y122OH, and D84, a unidentate ligand to the diferric cluster, previously identified in isolated β2, were observed in the α2β2 complex. These spectra also provide evidence for a conformational rearrangement at an additional β2 tyrosine(s), Y_<i>x</i>, in the α2β2/CDP/ATP complex. This study illustrates the utility of reaction-induced FT-IR spectroscopy in the study of complex enzymes