Heme-protein vibrational couplings in cytochrome c provide a dynamic link that connects the heme-iron and the protein surface

The active site of cytochrome c (Cyt c) consists of a heme covalently linked to a pentapeptide segment (Cys-X-X-Cys-His), which provides a link between the heme and the protein surface, where the redox partners of Cyt c bind. To elucidate the vibrational properties of heme c, nuclear resonance vibrational spectroscopy (NRVS) measurements were performed on 57Fe-labeled ferric Hydrogenobacter thermophilus cytochrome c 552, including 13C8-heme-, 13C 515N-Met-, and 13C15N-polypeptide (pp)-labeled samples, revealing heme-based vibrational modes in the 200- to 450-cm-1 spectral region. Simulations of the NRVS spectra of H. thermophilus cytochrome c552 allowed for a complete assignment of the Fe vibrational spectrum of the protein-bound heme, as well as the quantitative determination of the amount of mixing between local heme vibrations and pp modes from the Cys-X-XCys-His motif. These results provide the basis to propose that heme-pp vibrational dynamic couplings play a role in electron transfer (ET) by coupling vibrations of the heme directly to vibrations of the pp at the protein - protein interface. This could allow for the direct transduction of the thermal (vibrational) energy from the protein surface to the heme that is released on protein/protein complex formation, or it could modulate the heme vibrations in the protein/protein complex to minimize reorganization energy. Both mechanisms lower energy barriers for ET. Notably, the conformation of the distal Met side chain is fine-tuned in the protein to localize heme-pp mixed vibrations within the 250-to 400-cm-1 spectral region. These findings point to a particular orientation of the distal Met that maximizes ET

Optical spectroscopic studies of simple polyenes and complex xanthophylls

The optical spectroscopic properties of four polyenes, decatetraene, dodecapentaene, tetradecahexaene and hexadecaheptaene, two carotenes, β-carotene and lycopene, and five xanthophylls, neoxanthin, violaxanthin, lutein, zeaxanthin, and β-cryptoxanthin, were studied using steady-state absorption, fluorescence, fluorescence excitation or ultrafast transient absorption spectroscopy. High-performance liquid chromatography (HPLC) was carried out immediately prior to the spectroscopic experiments to obtain isomerically pure molecules. In the first part of the study, experiments on the polyenes were done at cryogenic temperatures in n-alkane mixed crystal solvents and provided enhanced spectral resolution compared to room temperature solutions and low-temperature glasses. This allowed discrimination of emission from cis and all-trans isomers. At 77 K, cis isomers of the polyenes displayed significantly more intense emission from the S1 (21A g-) state than observed for the all-trans isomers. In addition, the cis isomers exhibited spectral features associated with the forbidden S0 (11A g-) → S1 (21Ag -) transition. The effect of solvent polarizability on the S 1 (21Ag-) and S2 (11Bu+) energies of the all- trans polyenes was systematically evaluated. Upon extrapolation to zero polarizability, the values were found to be in close agreement with gas phase measurements. The experimental determinations were compared with theoretically-obtained values from the literature. In the second part of the study, spectral properties of the more complex carotenes and xanthophylls were investigated in solution using steady-state absorption spectroscopy in the visible and near-infrared (NIR) spectral regions. Also, cation radicals of the molecules were generated chemically and their spectra were interpreted using quantum computations. Finally, the steady-state and ultrafast transient absorption spectroscopic features of violaxanthin and zeaxanthin were investigated in solution, in pigment-protein complexes prepared from Photosystem II (PSII), and in isolated thylakoids. The goal of this part of the work was to pinpoint the location of zeaxanthin cation radical formation thought to be a component in excess energy dissipation in higher plants.

Optical spectroscopic studies of simple polyenes and complex xanthophylls

The optical spectroscopic properties of four polyenes, decatetraene, dodecapentaene, tetradecahexaene and hexadecaheptaene, two carotenes, β-carotene and lycopene, and five xanthophylls, neoxanthin, violaxanthin, lutein, zeaxanthin, and β-cryptoxanthin, were studied using steady-state absorption, fluorescence, fluorescence excitation or ultrafast transient absorption spectroscopy. High-performance liquid chromatography (HPLC) was carried out immediately prior to the spectroscopic experiments to obtain isomerically pure molecules. In the first part of the study, experiments on the polyenes were done at cryogenic temperatures in n-alkane mixed crystal solvents and provided enhanced spectral resolution compared to room temperature solutions and low-temperature glasses. This allowed discrimination of emission from cis and all-trans isomers. At 77 K, cis isomers of the polyenes displayed significantly more intense emission from the S1 (21A g-) state than observed for the all-trans isomers. In addition, the cis isomers exhibited spectral features associated with the forbidden S0 (11A g-) → S1 (21Ag -) transition. The effect of solvent polarizability on the S 1 (21Ag-) and S2 (11Bu+) energies of the all- trans polyenes was systematically evaluated. Upon extrapolation to zero polarizability, the values were found to be in close agreement with gas phase measurements. The experimental determinations were compared with theoretically-obtained values from the literature. In the second part of the study, spectral properties of the more complex carotenes and xanthophylls were investigated in solution using steady-state absorption spectroscopy in the visible and near-infrared (NIR) spectral regions. Also, cation radicals of the molecules were generated chemically and their spectra were interpreted using quantum computations. Finally, the steady-state and ultrafast transient absorption spectroscopic features of violaxanthin and zeaxanthin were investigated in solution, in pigment-protein complexes prepared from Photosystem II (PSII), and in isolated thylakoids. The goal of this part of the work was to pinpoint the location of zeaxanthin cation radical formation thought to be a component in excess energy dissipation in higher plants.

Theoretical and Spectroscopic Analysis of <i>N</i>,<i>N</i>′‑Diphenylurea and <i>N</i>,<i>N</i>′‑Dimethyl‑<i>N</i>,<i>N</i>′‑diphenylurea Conformations

Structural organization of macromolecules is highly dependent on the conformational propensity of the monomer units. Our goal is to systematically quantify differences in the conformational propensities of aromatic oligourea foldamer units. Specifically, we investigate the conformational propensities of <i>N</i>,<i>N</i>′-diphenylurea and <i>N</i>,<i>N</i>′-dimethyl-<i>N</i>,<i>N</i>′-diphenylurea in different media using a combination of theoretical methods, and infrared and nuclear magnetic resonance spectroscopies. Our results show variation in the conformational behavior upon adding methyl substituents on <i>N</i>,<i>N</i>′-diphenylurea, and varying the environments surrounding the compounds. Our energetic analyses and conformational distributions in the gas phase show predominance of the <i>cis</i>–<i><i>trans</i></i> and <i>trans</i>–<i>trans</i> conformations for <i>N</i>,<i>N</i>′-diphenylurea, while <i>cis</i>–<i>cis</i> conformation is favored for <i>N</i>,<i>N</i>′-dimethyl-<i>N</i>,<i>N</i>′-diphenylurea. In solution, our results support the <i>trans</i>–<i>trans</i> conformer as the predominant conformer for <i>N</i>,<i>N</i>′-diphenylurea, whereas the <i>cis</i>–<i>cis</i> and <i>cis</i>–<i>trans</i> forms are favored in <i>N</i>,<i>N</i>′-dimethyl-<i>N</i>,<i>N</i>′-diphenylurea. <i>N</i>,<i>N</i>′-Dimethyl-<i>N</i>,<i>N</i>′-diphenylurea also exhibits a more dynamic conformational behavior in solution, with constant fluctuations between <i>cis</i>–<i>cis</i> and <i>cis</i>–<i>trans</i> conformations. Our detailed quantitative analyses are an important aspect in fine-tuning desired conformations and dynamic properties of this class of oligomers by providing a molecular basis for the behavior at the monomeric level

Ligand Recruitment and Spin Transitions in the Solid-State Photochemistry of Fe^(III)TPPCl

We report evidence for the formation of long-lived photoproducts following excitation of iron­(III) tetraphenylporphyrin chloride (Fe^(III)TPPCl) in a 1:1 glass of toluene and CH₂Cl₂ at 77 K. The formation of these photoproducts is dependent on solvent environment and temperature, appearing only in the presence of toluene. No long-lived product is observed in neat CH₂Cl₂ solvent. A 2-photon absorption model is proposed to account for the power-dependent photoproduct populations. The products are formed in a mixture of spin states of the central iron­(III) metal atom. Metastable six-coordinate high-spin and low-spin complexes and a five-coordinate high-spin complex of iron­(III) tetraphenylporphyrin are assigned using structure-sensitive vibrations in the resonance Raman spectrum. These species appear in conjunction with resonantly enhanced toluene solvent vibrations, indicating that the Fe^(III) compound formed following photoexcitation recruits a toluene ligand from the surrounding environment. Low-temperature transient absorption (TA) measurements are used to explain the dependence of product formation on excitation frequency in this photochemical model. The six-coordinate photoproduct is initially formed in the high-spin Fe^(III) state, but population relaxes into both high-spin and low-spin state at 77 K. This is the first demonstration of coupling between the optical and magnetic properties of an iron-centered porphyrin molecule

Effects of Protein Structure on Iron–Polypeptide Vibrational Dynamic Coupling in Cytochrome <i>c</i>

Cytochrome <i>c</i> (Cyt <i>c</i>) has a heme covalently bound to the polypeptide via a Cys-X-X-Cys-His (CXXCH) linker that is located in the interface region for protein–protein interactions. To determine whether the polypeptide matrix influences iron vibrational dynamics, nuclear resonance vibrational spectroscopy (NRVS) measurements were performed on ⁵⁷Fe-labeled ferric <i>Hydrogenobacter thermophilus</i> cytochrome <i>c</i>-552, and variants M13V, M13V/K22M, and A7F, which have structural modifications that alter the composition or environment of the CXXCH pentapeptide loop. Simulations of the NRVS data indicate that the 150–325 cm^–1 region is dominated by N_His–Fe–S_Met axial ligand and polypeptide motions, while the 325–400 cm^–1 region shows dominant contributions from ν­(Fe–N_Pyr) (Pyr = pyrrole) and other heme-based modes. Diagnostic spectral signatures that directly relate to structural features of the heme active site are identified using a quantum chemistry-centered normal coordinate analysis (QCC-NCA). In particular, spectral features that directly correlate with CXXCH loop stiffness, the strength of the Fe–His interaction, and the degree of heme distortion are identified. Cumulative results from our investigation suggest that compared to the wild type (wt), variants M13V and M13V/K22M have a more rigid CXXCH pentapeptide segment, a stronger Fe–N_His interaction, and a more ruffled heme. Conversely, the A7F variant has a more planar heme and a weaker Fe–N_His bond. These results are correlated to the observed changes in reduction potential between wt protein and the variants studied here. Implications of these results for Cyt <i>c</i> biogenesis and electron transfer are also discussed