Optical Spectra of Fe(II) Cytochrome c Interpreted Using Molecular Dynamics Simulations and Quantum Mechanical Calculations (original) (raw)
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Journal of Physical Chemistry B, 2002
Porphyrin electronic transitions in heme proteins provide a useful tool for probing the protein environment, since the surrounding protein affects the porphyrin π-electron cloud. Perturbations can arise from structural distortions of the porphyrin ring, from the internal electric field generated by charged and polar groups, or from axial ligation to the heme iron. In this work, cytochrome c in aqueous solution or in glasses of trehalose or glycerol/water was examined as a function of temperature to evaluate the effect of fluctuations on the heme. The amide I band of cytochrome c in trehalose remains constant over a wide temperature excursion, indicating that interactions between the protein and the matrix do not change with temperature. The width of the Q(0,0) optical transition measured at low temperature (i.e., <100 K) reflects the temperature at which the glass was formed, while the temperature profiles of the widths for the protein in different solvents and glasses are similar at high temperature. The results were interpreted in terms of contributions from solvent-coupled and solvent-uncoupled motions. Molecular dynamics simulations of cytochrome c in explicit solvent were performed to investigate the structural distortions in the protein, and semiempirical quantum mechanics (Zindo/ S) was used to calculate the resultant changes in the spectroscopic transitions. A correlation between the calculated transition energies and the structural distortions in both the heme and the surrounding protein environment was observed and was invoked to characterize the origins of the temperature-dependent broadening of the electronic transitions seen in the visible spectra.
The Journal of Chemical Physics, 2005
We have measured and analyzed the low-temperature ͑T =10 K͒ absorption spectrum of reduced horse heart and yeast cytochrome c. Both spectra show split and asymmetric Q 0 and Q v bands. The spectra were first decomposed into the individual split vibronic sidebands assignable to B 1g ͑ 15 ͒ and A 2g ͑ 19 , 21 , and 22 ͒ Herzberg-Teller active modes due to their strong intensity in resonance Raman spectra acquired with Q 0 and Q v excitations. The measured band splittings and asymmetries cannot be rationalized solely in terms of electronic perturbations of the heme macrocycle. On the contrary, they clearly point to the importance of considering not only electronic perturbations but vibronic perturbations as well. The former are most likely due to the heterogeneity of the electric field produced by charged side chains in the protein environment, whereas the latter reflect a perturbation potential due to multiple heme-protein interactions, which deform the heme structure in the ground and excited states. Additional information about vibronic perturbations and the associated ground-state deformations are inferred from the depolarization ratios of resonance Raman bands. The results of our analysis indicate that the heme group in yeast cytochrome c is more nonplanar and more distorted along a B 2g coordinate than in horse heart cytochrome c. This conclusion is supported by normal structural decomposition calculations performed on the heme extracted from molecular-dynamic simulations of the two investigated proteins. Interestingly, the latter are somewhat different from the respective deformations obtained from the x-ray structures.
Journal of Physical Chemistry B, 2000
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.
The Journal of Physical Chemistry B, 2001
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.
Biochemistry, 2008
The oxidized state of cytochrome c is a subject of continuous interest, owing to the multitude of conformations which the protein can adopt in solution and on surfaces of artificial and cell membranes. The structural diversity corresponds to a variety of functions in electron transfer, peroxidase and apoptosis processes. In spite of numerous studies, a comprehensive analysis and comparison of native and nonnative states of ferricytochrome c has thus far not been achieved. This results in part from the fact that the influence of solvent conditions (i.e., ionic strength, anion concentration, temperature dependence of pH values) on structure, function and equilibrium thermodynamics has not yet been thoroughly assessed. The current study is a first step in this direction, in that it provides the necessary experimental data to compare different non-native states adopted at high temperature and alkaline pH. To this end, we employed visible electronic circular dichroism (ECD) and absorption spectroscopy to probe structural changes of the heme environment in bovine and horse heart ferricytochrome c as a function of temperature between 278 and 363 K at different neutral and alkaline pH values. A careful selection of buffers enabled us to monitor the partial unfolding of the native state at room temperature while avoiding a change to an alkaline state at high temperatures. We found compelling evidence for the existence of a thermodynamic intermediate of the thermal unfolding/folding process, termed III h , which is structurally different from the alkaline states, IV 1 and IV 2 , contrary to current belief. At neutral or slightly acidic pH, III h is populated in a temperature region between 320 and 345 K. The unfolded state of the protein becomes populated at higher temperatures. The ECD spectra of the B-bands of bovine and horse heart cytochrome c (pH 7.0) exhibit a pronounced couplet that is maintained below 343 K, before protein unfolding replaces it by a rather strong positive Cotton band. A preliminary vibronic analysis of the B-band profile reveals that the couplet reflects a B-band splitting of 350 cm -1 , which is mostly of electronic origin, due to the internal electric field in the heme cavity. Our results suggest that the conformational transition from the native state, III, into a thermally activated intermediate state, III h , does not substantially affect the internal electric field and causes only moderate rearrangements of the heme pocket, which involves changes, rather than a rupture, of the Fe 3+ -M80 linkage. In the unfolded state, as well as in the alkaline states IV and V, the band splitting is practically eliminated, but the positive Cotton effect observed for the B-band suggests that the proximal environment, encompassing H18 and the two cysteine residues 14 and 17, is most likely still intact and covalently bound to the heme chromophore. Both alkaline states IV and V were found to melt via intermediate states. Unfolded states probed at neutral and alkaline pH can be discriminated, owing to the different intensities of the Cotton bands of the respective B-band transitions. Differences between the ECD intensities of the B-bands of the different unfolded states and alkaline states most likely reflect different degrees of openness of the corresponding heme crevice.
Biophysical journal, 2007
We have measured the electronic circular dichroism (ECD) of the ferri-and ferro-states of several natural cytochrome c derivatives (horse heart, chicken, bovine, and yeast) and the Y67F mutant of yeast in the region between 300 and 750 nm. Thus, we recorded the ECD of the B-and Q-band region as well as the charge-transfer band at ;695 nm. The B-band region of the ferri-state displays a nearly symmetric couplet at the B 0 -position that overlaps with a couplet 790 cm ÿ1 higher in energy, which we assigned to a vibronic side-band transition. For the ferro-state, the couplet is greatly reduced, but still detectable. The B-band region is dominated by a positive Cotton effect at energies lower than B 0 that is attributed to a magnetically allowed iron/heme charge-transfer transition as earlier observed for nitrosyl myoglobin and hemoglobin. The Q-band region of the ferri-state is poorly resolved, but displays a pronounced positive signal at higher wavenumbers. This must result from a magnetically allowed transition, possibly from the methionine ligand to the d xy -hole of Fe 31 . For the ferro-state, the spectra resolve the vibronic structure of the Q v -band. A more detailed spectral analysis reveals that the positively biased spectrum can be understood as a superposition of asymmetric couplets of split Q 0 and Q v -states. Substantial qualitative and quantitative differences between the respective B-state and Q-state ECD spectra of yeast and horse heart cytochrome c can clearly be attributed to the reduced band splitting in the former, which results from a less heterogeneous internal electric field. Finally, we investigated the charge-transfer band at 695 nm in the ferri-state spectrum and found that it is composed of at least three bands, which are assignable to different taxonomic substates. The respective subbands differ somewhat with respect to their Kuhn anisotropy ratio and their intensity ratios are different for horse and yeast cytochrome c. Our data therefore suggests different substate populations for these proteins, which is most likely assignable to a structural heterogeneity of the distal Fe-M80 coordination of the heme chromophore.
Journal of Physical Chemistry B, 2000
Low-temperature UV-vis absorption and Stark-effect hole-burning spectra of Zn substituted cytochrome c are studied experimentally and theoretically using quantum mechanical and Poisson-Boltzmann electrostatics models. Both the Q and Soret bands show resolved splitting at temperatures below ∼180 K. The trend observed in the splittings when comparing cytochromes from different species is found to be the same as that observed for the Q(0,0) band of ferrous cytochrome c. The relative magnitudes of the Q and Soret splittings are found to be consistent with predictions based on Gouterman's four orbital model. For horse heart and yeast cytochrome c, which show the greatest difference in the UV-visible band splittings, Stark effect measurements on persistent spectral holes in the Q(0,0) band indicate that the protein-induced polarization is distinctly different for these two species. Incorporation of the protein electrostatic field as virtual point charges into quantum mechanical calculations utilizing the INDO/s semiempirical Hamiltonian is used to demonstrate that the effects of the protein on the heme electronic structure can be considerably different for the two proteins, consistent with the experimental observations.
Journal of Physical Chemistry B, 2000
Low-temperature UV-vis absorption and Stark-effect hole-burning spectra of Zn substituted cytochrome c are studied experimentally and theoretically using quantum mechanical and Poisson-Boltzmann electrostatics models. Both the Q and Soret bands show resolved splitting at temperatures below ∼180 K. The trend observed in the splittings when comparing cytochromes from different species is found to be the same as that observed for the Q(0,0) band of ferrous cytochrome c. The relative magnitudes of the Q and Soret splittings are found to be consistent with predictions based on Gouterman's four orbital model. For horse heart and yeast cytochrome c, which show the greatest difference in the UV-visible band splittings, Stark effect measurements on persistent spectral holes in the Q(0,0) band indicate that the protein-induced polarization is distinctly different for these two species. Incorporation of the protein electrostatic field as virtual point charges into quantum mechanical calculations utilizing the INDO/s semiempirical Hamiltonian is used to demonstrate that the effects of the protein on the heme electronic structure can be considerably different for the two proteins, consistent with the experimental observations.
Biophysical Journal, 1999
We 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.