Spectral Splitting in the α ( Q 0,0 ) Absorption Band of Ferrous Cytochrome c and Other Heme Proteins † (original) (raw)
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.
European Journal of Biochemistry, 1976
Tin (Sn4+) and zinc (Zn") derivatives of horse heart cytochrome c have been prepared and their optical spectra have been characterized. Zinc cytochrome c has visible absorption maxima at 549 and 585 nm and Soret absorption at 423 nm. Tin cytochrome c shows visible absorption maxima at 536 and 574 nm and Soret absorption at 410 nm. Unlike iron cytochrome c in which the emission spectrum of the porphyrin is almost completely quenched by the central metal, the zinc and tin derivatives of cytochrome c are both fluorescent and phosphorescent. The fluorescence maxima of zinc cytochrome c are at 590 and 640 nm and the fluorescence lifetime is 3.2 ns. The fluorescence maxima of Sn cytochrome are at 580 and 636 nm and the fluorescence lifetime is under 1 ns. The quantum yield of fluorescence is Zn > Sn while the quantum yield of phosphorescence is Sn > Zn. At 77 K the fluorescence and phosphorescence emission spectra of Sn and Zn cytochrome c show evidence of resolution into vibrational bands. The best resolved bands occur at frequency differences 750 cm-' and 1540-1550 cm-' from the 0-0 transition. These frequencies correspond with those obtained by resonance Raman spectroscopy for in-plane deformations of the porphyrin macrocycle.
RESOLVED FLUORESCENCE EMISSION SPECTRA OF IRON-FREE CYTOCHROME c
Photochemistry and Photobiology, 1982
The fluorescence emission of iron-free cytochrome c ('Cyt c ) in a glassy matrix was investigated under conditions of very low temperature (4.2 K) and narrow bandwidth laser excitation. Excitation into the vibronic band, QJl.0) resulted in highly resolved emission spectra of linewidth 1&20cm-'. Using the model of selective excitation developed by Abram et a/. (1975) and McColgan et a/. (1978), the emission spectra of vibronic excitation afforded a method to investigate excited state vibrational structure. Furthermore, emission profiles have shown that in 'Cyt c, the site distribution (inhomogeneous broadening) has a width in the order of 200cm-'.
Evidence for a band III analogue in the near-infrared absorption spectra of cytochrome oxidase
Biochemical and Biophysical Research Communications, 1992
Ground state near-infrared absorption spectra of fully reduced unliganded and fully reduced CO @+ CUA+ g2+-CO CUB+) cymchrome E oxidase were investigated. Flash-photolysis time-resolved absorption difference spectra of the mixed-valence h3+ CUA~+ a32+-C0 CUB+) and the fully reduced CO complexes were also studied. A band near 785 nm (E -50 M-km-l) was observed in the fully reduced unliganded enzyme and the CO photoproducts. The time-resolved 785 nm band disappeared on the same timescale (tin -7 ms) as CO recombined with cytochrome a32+. This band, which is attributed to the unliganded five coordinate ferrous cytochrome a2+, has some characteristics of band III in deoxy-hemoglobin and deoxy-myoglobin. A second band was observed at -710 nm (E -80 M-km-l) in the fully reduced unliganded and the fully reduced CO complexes. This band, which we assign to the low spin ferrous cytochrome 3, appears to be affected by the ligation state at the cytochrome a32+ site. 0 1992 Academic Press, 1°C.
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.
Fluorescence line narrowed spectra of Zn and metal-free cytochrome c
Journal of Fluorescence, 1991
Fluorescence line narrowing (FLN) spectroscopy was used to study the role of the polypeptide chain in influencing the spectrum of Zn-substituted cytochrome c (Zn cyt c) and metal-free cyt c (porphyrin cyt c). For both derivatives the spectra show characteristics of relaxed fluorescence from an inhomogeneously broadened sample. Zero phonon lines and phonon wings can be clearly distinguished, and vibrational frequencies of the ground and excited states were identified. The inhomogeneous distribution width for porphyrin cyt c is slightly wider than that of Zn cyt c and a second population of molecules was apparent in the porphyrin cyt c. The phonon coupling was greater for Zn cyt c than for porphyrin cyt c, which may he due to the extra coupling to the polypeptide chain by metal ligation.