A resonance Raman enhancement mechanism for axial vibrational modes in the pyridine adduct of myoglobin proximal cavity mutant (H93G).

The proximal cavity mutant of myoglobin consists of a mutation of the proximal histidine to glycine (H93G), which permits exogenous ligands to bind to the heme iron. A non-native pyridine ligand can ligate to the heme to yield a five-coordinate adduct, H93G(Pyr), that cannot be formed freely in solution since the six-coordinate bis-pyridine adduct is more stable than the five-coordinate adduct. We have used resonance Raman spectroscopy in the Soret band region of the heme to study the enhancement of axial vibrations of bound pyridine in the H93G(Pyr) adduct. The observation that the pyridine ring breathing mode (ν(1)) and the symmetric ring stretching (ν(3)) modes are enhanced under these conditions is explained by a computational approach that shows that coupling of the π-system of the heme with the p-orbitals of the pyridine is analogous to π-backbonding in diatomic ligand adducts of heme proteins. The result has the broader significance that it suggests that the resonance enhancement of pyridine modes could be an important aspect of Raman scattering of pyridine on conducting surfaces such as those studied in surface enhanced Raman scattering experiments.

Kinetic analysis of a naturally occurring bioremediation enzyme: dehaloperoxidase-hemoglobin from Amphitrite ornata.

The temperature dependence of the rate constant for substrate oxidation by the dehaloperoxidase-hemoglobin (DHP) of Amphitrite ornata has been measured from 278 to 308 K. The rate constant is observed to increase over this range by approximately a factor of 2 for each 10 °C temperature increment. An analysis of the initial rates using a phenomenological approach that expresses the peroxidase ping-pong mechanism in the form of the Michaelis-Menten equation leads to an interpretation of the effects in terms of the fundamental rate constants. The analysis of kinetic data considers a combination of diffusion rate constants for substrate and H(2)O(2), elementary steps involving activation and heterolysis of the O-O bond of H(2)O(2), and two electron transfers from the substrate to the iron. To complete the analysis from the perspective of turnover of substrate into product, density function theory (DFT) calculations were used to address the fate of phenoxy radical intermediates. The analysis suggests a dominant role for diffusion in the kinetics of DHP.

Excited-state geometry method for calculation of the absolute resonance Raman cross sections of the aromatic amino acids.

The time correlator formalism was used to calculate the absolute resonance Raman cross sections for the aromatic amino acids based on density functional theory calculations of the ground-state potential energy surfaces combined with projection along normal mode eigenvectors in the excited state. The geometric difference between the minima of the ground and excited states along each normal mode was calculated to provide inputs for the time correlator in the linear approximation. The calculated dimensionless nuclear displacements, Delta(i), provide the electron-phonon coupling constants, S(i) = Delta(i)(2)/2, for the corresponding Raman active mode of frequency omega(t). The method is generally applicable to molecules that are Franck-Condon active. As an example we have chosen to calculate the absolute resonance Raman cross sections of models of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. We discuss the role played by substituents on the aromatic ring that decrease vibronic activity to a level that permits application of the time correlator. While the method may have limitations for molecules of high symmetry, the current study of excited-state displacements and electronic structure indicates that the L(a),(b) states are Franck-Condon active in the aromatic molecules studied.

Preparation of protein gradients through the controlled deposition of protein-nanoparticle conjugates onto functionalized surfaces.

This paper describes a simple method for the preparation and characterization of protein density gradients on solid supports. The method employs colloidal metal nanoparticles as protein carriers and optical tags and is capable of forming linear, exponential, 1D, 2D, and multiprotein gradients of varying slope without expensive or sophisticated surface patterning techniques. Surfaces patterned with proteins using the procedures described within are shown to support cell growth and are thus suitable for studies of protein-cell interactions.