Spectral Splitting in the α ( Q 0,0 ) Absorption Band of Ferrous Cytochrome c and Other Heme Proteins † (original) (raw)
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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.
The Journal of Physical Chemistry B, 2006
Cytochrome c (Cyt c) is a heme protein involved in electron transfer and also in apoptosis. Its heme iron is bisaxially ligated to histidine and methionine side chains and both ferric and ferrous redox states are physiologically relevant, as well as a ligand exchange between internal residue and external diatomic molecule. The photodissociation of internal axial ligand was observed for several ferrous heme proteins including Cyt c, but no time-resolved studies have been reported on ferric Cyt c. To investigate how the oxidation state of the heme influences the primary photoprocesses, we performed a comprehensive comparative study on horse heart Cyt c by subpicosecond time-resolved resonance Raman and femtosecond transient absorption spectroscopy. We found that in ferric Cyt c, in contrast to ferrous Cyt c, the photodissociation of an internal ligand does not take place, and relaxation dynamics is dominated by vibrational cooling in the ground electronic state of the heme. The intermolecular vibrational energy transfer was found to proceed in a single phase with a temperature decay of ∼7 ps in both ferric and ferrous Cyt c. For ferrous Cyt c, the instantaneous photodissociation of the methionine side chain from the heme iron is the dominant event, and its rebinding proceeds in two phases, with time constants of ∼5 and ∼16 ps. A mechanism of this process is discussed, and the difference in photoinduced coordination behavior between ferric and ferrous Cyt c is explained by an involvement of the excited electronic state coupled with conformational relaxation of the heme.
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, 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 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.
Journal of Inorganic Biochemistry, 1981
The K-absorption edges of the heme iron have been determined from the x-ray absorption spectra of native (Nat) and carboxymethylated (Cm) cytochrome c. The data collected at pH 6.5 and 10 and in both the oxidation states of the hemes indicate that the latter parameter is the one responsiile for the observed chemical shifts of the absorption edges in both the cytochromes The implications regarding a proposed structural model for Cm cytochrome c are discussed_ Abbreviations Nat cyt c, cytochrome c in the native form; Cm cyt c, carboxymethylated cytochrome c.
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.