Abstract
Resonance Raman spectra have been recorded from ferri-cytochromec bound to phospholipid vesicles composed of dimyristoyl phosphatidylglycerol (DMPG), dioleoyl phosphatidylglycerol (DOPG) or dioleoyl phosphatidylglycerol-dioleoyl phasphatidylcholine (DOPG-OPC) (70 : 30 mole/mole). Lipid binding induces very significant conformational changes in the protein molecule. The resonance Raman spectra differ in their content of bands originating from two different conformational species, I and II, of the protein, and from two different spin and coordination states of the heme in conformation II. Data of sufficiently high precision were obtained that the spectra of the individual species could be quantitated by a constraint interative fitting routine using single Lorentzian profiles. In the high frequency, or marker band region (1200 to 1700 cm−1), the frequencies, half widths and relative intensities of the individual bands could be estimated from previous surface enhanced resonance Raman measurements on cytochromec adsorbed on a silver electrode. These were then further optimized to yield both the spectral parameters and relative contents of the different species. In the low frequency, or finger-print, region (200 to 800 cm−1), the spectral parameters of the individual species were obtained from difference spectra derived by sequential subtraction between the spectra of ferri-cytochromec in the three different lipid systems, using the relative proportions of the species derived from the marker band region. These parameters were then subsequently refined by iterative optimization. The optimized spectral parameters in both frequency regions for the six-coordinated low spin states I and II, and for the five-coordinated high spin state II are presented. The proportion of state II, in which hence the heme crevice assumes an open structure, and of the five-coordinated high spin configuration, is found to increase on binding ferri cytochromec to negatively charged lipid vesicles. The extent of this conformational change increases in the order: DOPG-DOPC<DOPG<DMPG, with a parallel decrease of the proportion of the conformational state I, whose structure is similar to that of the uncomplexed ferri-cytochrome c in solution. Similar conformational changes are found for ferro-cytochromec compared to those obtained with the oxidized species on binding to lipids. The present work is essential for studies which seek to analyze, in any detailed fashion, the conformational transitions in the heme protein which take place in response to changes in the lipid environment.
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References
Alshuth T (1985) Kinetische und strukturelle Untersuchungen am Chromophor von Bacteriorhodopsin mit Hilfe zeitaufgelöster Resonanz-Raman-Spektroskopie. P.h.D. Thesis, Universität Göttingen
Azzi A, Fleischer S, Chance B (1969) Cytochromec — phospholipid interactions. Biochem Biophys Res Commun 36:322–327
Brautigan DL, Ferguson-Miller S, Margoliash E (1978) Mitochondrial cytochromec: Preparation and activity of native and chemically modified cytochromesc. Methods Enzymol 53D:128–164
Choi S, Spiro TG (1983) Out-of-plane deformation modes in the resonance Raman spectra of metalloporphyrins and heme proteins. J Am Chem Soc 105:3683–3692
Cotton TM, Schultz SG, Van Duyne RP (1980) Surface enhanced resonance Raman scattering from cytochromec and myoglobin adsorbed on a silver electrode. J Am Chem Soc 102:7960–7962
Devaux PF, Hoatson GL, Favre E, Fellmann P, Farren B, MacKay AL, Bloom M (1986) Interaction of cytochromec with mixed dimyristoylphosphatidylcholine-dimyristoylphosphatidylserine bilayers: a deuterium nuclear magnetic resonance study. Biochemistry 25:3804–3812
Fraser RDB, Suzuki E (1970) Biological applications. In: Blackburn JA (ed) Spectral analysis: methods and techniques. Dekker, New York pp 171–211
de Groot J, Hester RE (1987) Surface enhanced resonance Raman spectroscopy of oxyhemoglobin adsorbed onto colloidal silver. J Phys Chem 91:1693–1696
Heimburg T (1989) Untersuchungen der physikalischen Eigenschaften von biologischen- und Modellmembranen. P.h.D. Thesis, Universität Göttingen
Hildebrandt P, Stockburger M (1989a) Cytochromec at charged interfaces: 1. Conformational and redox equilibria at the electrode/electrolyte interface probed by surface enhanced resonance Raman spectroscopy. Biochemistry 28:6710–6721
Hildebrandt P, Stockburger M (1989b) Cytochromec at charged interfaces: 2. Complexes with negatively charged macromolecular systems studied by resonance Raman spectroscopy. Biochemistry 28:6722–6728
Kimelberg HK, Lee CP, Claude A, Mrena E (1970) Interactions of cytochromec with phospholipid membranes. J Membr Biol 2:235–251
Kitagawa T, Ozaki Y (1987) Infrared and Raman spectra of metalloporphyrins. Struct Bonding 64:71–114
Lee H, Kitagawa T, Abe M, Pandey RK, Leung HK, Smith KM (1986) Characterization of low frequency resonance Raman bands of metalloporphyrin IX. Observation of isotope shifts and normal coordinate treatments. J Mol Struc 146:329–347
Maddams FW (1980) The scope and limitations of curve fitting. Appl Spectrosc 34:245–267
Niki K, Kawasaki Y, Kimura Y, Higuchi Y, Yasuoka N (1989) Surface enhanced Raman scattering of cytochromes c3 adsorbed on silver electrode and their redox behavior. Langmuir 3:982–986
Parthasarathi N, Hansen C, Yamaguchi S, Spiro TG (1987) Metalloporphorin core size resonance Raman marker bands revisited: Implications for the interpretation of hemoglobin photoproduct Raman frequencies. J Am Chem Soc 109:3865–3871
Peschke J, Möhwald H (1987) Cytochromec interaction with phospholipid monolayers and vesicles. Coll Surf 27:305–326
Rousseau DL (1981) Raman difference spectroscopy as a probe of biological molecules. J Raman Spectrosc 10:94–99
Smulevich G, Spiro TG (1985) Surface enhanced Raman spectroscopic evidence that adsorption on silver particles can denature heme proteins. J Phys Chem 89:5168–5173
Spiro TG (1983) The resonance Raman spectroscopy of metalloporphyrins and heme proteins. In: Lever APB, Gray HB (eds) Iron porphyrins, II. Addison-Wesley, Reading, pp 89–159
Verma AL, Kimura K, Yagi T, Nakamura A, Inokuchi H, Kitagawa T (1989) SERR evidence for enzymatic reduction of cytochrome c3 adsorbed on Ag colloids. Chem Phys Lett 159:189–192
Wolf CR, Miles JS, Seilman S, Burke MD, Rospendowski BN, Kelly K, Smith WE (1988) Evidence that the catalytic differences of two structurally homologous forms of cytochrome P-450 relate to their heme environments. Biochemistry 27:1597–1603
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Hildebrandt, P., Heimburg, T. & Marsh, D. Quantitative conformational analysis of cytochromec bound to phospholipid vesicles studied by resonance Raman spectroscopy. Eur Biophys J 18, 193–201 (1990). https://doi.org/10.1007/BF02427378
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DOI: https://doi.org/10.1007/BF02427378