Sampling Field Heterogeneity at the Heme of c-Type Cytochromes by Spectral Hole Burning Spectroscopy and Electrostatic Calculations

AbstractWe report on a comparative investigation of the heme pocket fields of two Zn-substituted c-type cytochromes—namely yeast and horse heart cytochromes c—using a combination of hole burning Stark spectroscopy and electrostatic calculations. The spectral hole burning experiments are consistent with different pocket fields experienced at the hemes of the respective cytochromes. In the case of horse heart Zn-cytochrome c, two distinguishable electronic origins with different electrostatic properties are observed. The yeast species, on the other hand, displays a single electronic origin. Electrostatic calculations and graphics modeling using the linearized finite-difference Poisson-Boltzmann equation performed at selected time intervals on nanosecond-molecular dynamics trajectories show that the hemes of the respective cytochromes sample different potentials as they explore conformational space. The electrostatic potentials generated by the protein matrix at the heme show different patterns in both cytochromes, and we suggest that the cytochromes differ by the number of “electrostatic substates” that they can sample, thus accounting for the different spectral populations observed in the two cytochromes

Water Structure Changes Induced by Hydrophobic and Polar Solutes Revealed by Simulations and Infrared

A combination of simulations and Fourier transform infrared spectroscopy was used to examine the effect of three ionic solutes ͑KCl, NaCl, and KSCN͒, the polar solute urea, and the osmolyte trimethylamine-N-oxide ͑TMAO͒ on a water structure. The ionic solutes increase the mean waterwater H-bond angle in their first hydration shell concomitantly shifting the OH stretching mode to higher frequency, and shifting the HOH bending mode to lower frequency. TMAO decreases the mean water-water H-bond angle in its first hydration shell, shifts the OH stretching mode frequency down, and shifting the HOH bending mode frequency up. Urea has no effect on the mean H-bond angle, OH stretch, and HOH bend frequencies. These results can be explained in terms of changes in the relative proportions of two H-bond angle populations: Ionic solutes increase the population of more distorted ͑larger angle͒ H bonds relative to the less distorted population, TMAO has the reverse effect, while urea does not affect the H-bond angle probability distribution. The negligible effect of urea on water structure supports the direct binding model for urea-induced protein denaturation

Water Structure Changes Induced by Hydrophobic and Polar Solutes Revealed by Simulations and Infrared

A combination of simulations and Fourier transform infrared spectroscopy was used to examine the effect of three ionic solutes ͑KCl, NaCl, and KSCN͒, the polar solute urea, and the osmolyte trimethylamine-N-oxide ͑TMAO͒ on a water structure. The ionic solutes increase the mean waterwater H-bond angle in their first hydration shell concomitantly shifting the OH stretching mode to higher frequency, and shifting the HOH bending mode to lower frequency. TMAO decreases the mean water-water H-bond angle in its first hydration shell, shifts the OH stretching mode frequency down, and shifting the HOH bending mode frequency up. Urea has no effect on the mean H-bond angle, OH stretch, and HOH bend frequencies. These results can be explained in terms of changes in the relative proportions of two H-bond angle populations: Ionic solutes increase the population of more distorted ͑larger angle͒ H bonds relative to the less distorted population, TMAO has the reverse effect, while urea does not affect the H-bond angle probability distribution. The negligible effect of urea on water structure supports the direct binding model for urea-induced protein denaturation

Spectral analysis of cytochrome c: effect of heme conformation, axial ligand, peripheral substituents and local electric fields,

We present in this work low-temperature visible absorption spectra for recombinant Thermus thermophilus cytochrome c 552 . The Q-band presents a remarkable splitting at low temperature. We performed quantum chemical calculations to evaluate quantitatively the effect of heme conformation, axial ligand, peripheral substituents and local electric fields on the electronic spectra. In an attempt to find correlation between protein structure and spectral splitting, we carried out the same calculations on three other cytochrome c's: horse heart, tuna heart, and yeast. The quantum chemical calculations were performed at the INDO level with extensive configuration interaction. The electric field at the heme pocket was included in the calculations through a set of point charges fitting the actual electric field. The results obtained show clearly that all mentioned effects contribute to the observed spectral splitting in a complex nonadditive way