Proc. Natl. Acad. Sci. USA
Vol. 76, No. 8, pp. 3865-3869, August 1979 Biophysics
Protein influence on the heme in cytochrome c: Evidence from Raman difference spectroscopy (electronic structure/vibrational modes/porphyrins)
J. A. SHELNUTTtf, D. L. ROUSSEAUt§, JUDY K.
DETHMERS1, AND E. MARGOLIASHT
tBell Laboratories, Murray Hill, New Jersey 07974; and IDepartment of Biochemistry and Molecular Biology, Northwestern University, Evanston, Illinois 60201
Contributed by Emanuel Margoliash, May 17, 1979
Raman difference spectra have been obtained ABSTRACT for the cytochromes c of a number of species by simultaneous data acquisition from two samples. Frequency differences as small as 0.1 cm-l can be measured reproducibly by the technique we have developed. In comparisons between cytochromes c isolated from two different species, the frequency differences in the heme vibrational modes range from 0 to 6 cm-l. The vibrational frequencies of the heme are sensitive to the electronic charge density on the porphyrin macrocycle. The frequency differences are interpreted in terms of the influence of the heme-packed aromatic and highly electronegative amino acid side chains on the 7r* charge density and distribution on the heme. Such a control of the electronic properties of the heme by the protein may be important for the function of cytochrome C.
The relationship between protein structure and biological function in cytochrome c has been extensively investigated (1-3). An important aspect of this relationship is the interaction between the heme prosthetic group and the protein. An understanding of this interaction can be approached by comparative studies of the proteins of different species because the amino acid sequences of over 100 different cytochromes c have been determined (4, 5). However, the physical techniques that can examine the protein-heme interaction are very limited. Electron spin resonance (see ref. 6) can probe only ferric cytochrome c because the ferrous protein has a spin of zero. Optical absorption bands in the heme region are broad and only very small changes in the spectrum are observed (7) with substantial changes in amino acid sequence. Circular dichroism studies (8) of the heme region do show protein-dependent effects, but the complexity of the contributions to the spectra makes interpretation difficult. In NMR spectra heme resonances are found to vary only with large changes in the protein environment (9), although NMR studies (10) of the proteins of various species has allowed some comparisons of the near heme environments to be made. In contrast, resonance Raman spectroscopy should offer the proper richness, sensitivity, and resolution for a detailed examination of protein-dependent effects on the prosthetic group. Because of resonance enhancement, the spectra contain only vibrational modes of the heme and are free of complicating features due to either the protein or the solvents used (11). The frequencies of the porphyrin ring vibrational modes are sensitive to changes in the force field that result from changes in the electronic structure of the heme. In particular, appropriate amino acid sequence differences in heme proteins might be expected to affect the electron density in molecular orbitals of the porphyrin moiety, bringing about differences in the force constants of the normal modes and the geometry of the nuclei of the heme. To date, no variations in'the frequencies of the
normal modes of the heme have been reported for a homologous series of cytochromes, although differences in the normal mode frequencies have been observed upon changes in the iron ligation (12, 13) and spin state (14, 15). Moreover, the protein-dependent frequency shifts of Raman lines may be small and the ability to detect small shifts in broad lines (5-10 cm-1) is limited to about 1-2 cm-1 with conventional techniques. This is not the case for Raman difference spectroscopy (RDS). The RDS technique we have developed is readily capable of detecting differences of down to 10-2 of the full width of the lines seen in cytochrome c, so that differences as small as 0.1 cm-1 can be reliably measured. Comparisons of the cytochromes c of several species suggest that the Raman frequency differences are due to interactions of the heme with different amino acid side chains in the near heme environment. MATERIALS AND METHODS Horse, tuna, pigeon, dog, and Candida krusei cytochromes c were purchased from Sigma. Rhodospirillum rubrum cytochrome C2 was prepared according to Sponholtz et al. (16). Iso-i and iso-2 cytochromes c from bakers' yeast were prepared according to Sherman et al. (17), and spider monkey, human, cow, turtle, and horse cytochromes c were prepared from heart tissue by the procedure of Margoliash and Walasek (18) as modified by Brautigan et al. (19). Before spectra were taken all preparations were filtered through a column of Sephadex G-50 superfine (Pharmacia) to completely separate polymeric material, which caused a characteristic background fluorescence and a decrease in sensitivity of the difference spectra resulting from the increase in noise level. In general, the spectra were obtained with ferrocytochrome c (minimal dithionite reduced) at a concentration of about 0.2 mM, buffered in 10 or 25 mM Tris acetate, pH 7.8. With the yeast iso-i cytochrome, a trace of mercaptoethanol was added to prevent disulfide dimer formation at cysteine-102. Spectra were obtained in Pyrex or quartz cells at room temperature, taking from 2 to 10 hr for completion. At the end of each run there were no changes in the Raman spectra that could be attributed to the formation of ferricytochrome c. On several occasions optical spectra were taken at the end of the run to further demonstrate that no autooxidation had occurred. Gel filtration before and after a run confirmed that no polymeric material was present or had formed. Raman difference spectra were obtained by a modification of the technique reported by Kiefer (20, 21) and will be described in detail elsewhere. Briefly, samples were placed in a cylindrical cell with a partition along a diameter such that the Abbreviation: RDS, Raman difference spectroscopy.
§ To whom correspondence should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
t On leave from School of Physics, Georgia Institute of Teghnology,
Atlanta, GA 30332; permanent address; Sandia Laboratories, D5114, Albuquerque, NM 87185.
3865
3866
Biophysics: Shelnutt et al.
two samples were alternately illuminated by the argon ion laser at 5287 A as the cell was rotated. Thus both samples could be examined for each frequency setting of the monochromator. As the cell was rotated a synchronous signal was sent to gating and counting electronics to allow independent accumulation of the data from each sample and subsequent independent storage in a Texas Instruments 960B computer. The spectral slit width was 2 cm-' and the spectrometer was advanced in steps ranging from 0.1 to 0.5 cm-1. The spectra were scanned 10-25 times to signal average the data, and up to 4096 points could be collected for each of the two spectra. Under these experimental conditions the Raman lines of cytochromes c display their natural linewidths. To obtain a difference spectrum the intensity of a Raman line in one spectrum was adjusted to equal the intensity of that line in the other spectrum and the two spectra were subtracted digitally. The difference spectrum of two closely spaced and equally intense Lorentzian lines has the shape of a derivative of the line. For small frequency differences, Av, between the two Lorentzians, the frequency difference (20-22) between the maximum and the minimum in the difference spectrum is larger (r/v/). If r is the full width at half height of the Lorentzian lines of peak intensity, I, then the intensity (peakto-valley) in the difference spectrum, Io, is related to Av by Io = 2.6IoAv/r. By using this relationship for small frequency differences and calculating a calibration curve for larger differences, the size of the frequency shift, Av, may be obtained from the intensity in the difference spectrum, provided the intensity of the Raman line in the two materials being compared is balanced. This balance is achieved by multiplying the intensity of one spectrum by a constant (balance ratio) prior to calculating the difference spectrum of that particular line. With this RDS technique very small frequency differences may be detected, because errors due to thermal drift and irreproducibility of the monochromator are eliminated by obtaining data for both samples at each setting of the spectrometer. The spectral resolution and the spectrometer frequency stepping interval that we used in this study have been shown from model calculations to introduce no larger than a 6% error in the frequency differences.
Proc. Natl. Acad. Sci. USA 76 (1979)
Frequency,
cm-1
FIG. 1. At the top, Raman difference spectrum of tuna cytochrome c versus horse cytochrome c. For these data the spectrometer was advanced in 0.1 cm-' increments. At the bottom, Raman spectra of tuna and horse ferrocytochromes c. The 750 cm-' line of tuna cytochrome c is 0.1 cm-1 higher in frequency than that of the horse protein.
vidual vibrational modes independently and thereby determine whether frequency differences exist. This is particularly important for samples in which the relative intensity of the Raman lines differ for the two proteins compared. For example, the R. rubrum cytochrome C2 difference spectrum with a balance ratio of 0.85 shows clear differences in the 1127, 1310, and 1584
I. c 0
c
RESULTS Ferrocytochrome c has absorption bands in the red-yellow region, from which strong resonance Raman scattering takes place (11). There is an absorption band near 5500 A (a band), which is a zero vibrational level transition, and a broad vibrational side band (f3 band) centered at about 5200 A. Excitation into the a band results in general enhancement of all the symmetry-allowed in-plane porphyrin modes, whereas excitation in the 3 band preferentially enhances the vibrational modes responsible for that portion of the vibrational side band. The report examines data obtained with excitation in the : band at 5287 A. This excitation wavelength results in enhancement of vibrational modes in the 700-1600 cm-1 region. Spectra of the 750 cm-1 line in horse and tuna cytochromes c are shown in Fig. 1. Although the frequency difference in this line for the two proteins is only 0.1 cm-1, it can be reproducibly determined from the RDS spectrum. Fig. 2 gives the difference spectra between horse cytochrome c and that of pigeon, human, bakers' yeast iso-i, and the cytochrome C2 of R. rubrum. The balance factor, by which the horse cytochrome c spectrum was multiplied to obtain each difference spectrum, is indicated in the figure. These factors were selected so as to balance indi-
c
E.a
cr
412
750
Horse
825a
851127
800
1000
Frequency,
1310
1200
14
cm'
FIG. 2. Raman difference spectra between a series of cytochromes and the horse protein. For these data the spectrometer was advanced in 0.5 cm-' increments. The Raman spectrum of horse ferrocytochrome c is shown at the bottom. The scale factor for each difference spectrum is indicated on the left. The balance factor by which the horse cytochrome c data were multiplied is indicated in the label for each difference spectrum. (Inset) Difference spectrum of the R. rubrum protein at a balance ratio of 1.4, for which the frequency difference for the 750 cm-1 line becomes apparent. c
Biophysics: Shelnutt et al.
cm-1 lines, while with a ratio of 1.4 the difference in the 750 cm-1 line becomes evident (see Fig. 2). There are no observable differences (
Vol. 76, No. 8, pp. 3865-3869, August 1979 Biophysics
Protein influence on the heme in cytochrome c: Evidence from Raman difference spectroscopy (electronic structure/vibrational modes/porphyrins)
J. A. SHELNUTTtf, D. L. ROUSSEAUt§, JUDY K.
DETHMERS1, AND E. MARGOLIASHT
tBell Laboratories, Murray Hill, New Jersey 07974; and IDepartment of Biochemistry and Molecular Biology, Northwestern University, Evanston, Illinois 60201
Contributed by Emanuel Margoliash, May 17, 1979
Raman difference spectra have been obtained ABSTRACT for the cytochromes c of a number of species by simultaneous data acquisition from two samples. Frequency differences as small as 0.1 cm-l can be measured reproducibly by the technique we have developed. In comparisons between cytochromes c isolated from two different species, the frequency differences in the heme vibrational modes range from 0 to 6 cm-l. The vibrational frequencies of the heme are sensitive to the electronic charge density on the porphyrin macrocycle. The frequency differences are interpreted in terms of the influence of the heme-packed aromatic and highly electronegative amino acid side chains on the 7r* charge density and distribution on the heme. Such a control of the electronic properties of the heme by the protein may be important for the function of cytochrome C.
The relationship between protein structure and biological function in cytochrome c has been extensively investigated (1-3). An important aspect of this relationship is the interaction between the heme prosthetic group and the protein. An understanding of this interaction can be approached by comparative studies of the proteins of different species because the amino acid sequences of over 100 different cytochromes c have been determined (4, 5). However, the physical techniques that can examine the protein-heme interaction are very limited. Electron spin resonance (see ref. 6) can probe only ferric cytochrome c because the ferrous protein has a spin of zero. Optical absorption bands in the heme region are broad and only very small changes in the spectrum are observed (7) with substantial changes in amino acid sequence. Circular dichroism studies (8) of the heme region do show protein-dependent effects, but the complexity of the contributions to the spectra makes interpretation difficult. In NMR spectra heme resonances are found to vary only with large changes in the protein environment (9), although NMR studies (10) of the proteins of various species has allowed some comparisons of the near heme environments to be made. In contrast, resonance Raman spectroscopy should offer the proper richness, sensitivity, and resolution for a detailed examination of protein-dependent effects on the prosthetic group. Because of resonance enhancement, the spectra contain only vibrational modes of the heme and are free of complicating features due to either the protein or the solvents used (11). The frequencies of the porphyrin ring vibrational modes are sensitive to changes in the force field that result from changes in the electronic structure of the heme. In particular, appropriate amino acid sequence differences in heme proteins might be expected to affect the electron density in molecular orbitals of the porphyrin moiety, bringing about differences in the force constants of the normal modes and the geometry of the nuclei of the heme. To date, no variations in'the frequencies of the
normal modes of the heme have been reported for a homologous series of cytochromes, although differences in the normal mode frequencies have been observed upon changes in the iron ligation (12, 13) and spin state (14, 15). Moreover, the protein-dependent frequency shifts of Raman lines may be small and the ability to detect small shifts in broad lines (5-10 cm-1) is limited to about 1-2 cm-1 with conventional techniques. This is not the case for Raman difference spectroscopy (RDS). The RDS technique we have developed is readily capable of detecting differences of down to 10-2 of the full width of the lines seen in cytochrome c, so that differences as small as 0.1 cm-1 can be reliably measured. Comparisons of the cytochromes c of several species suggest that the Raman frequency differences are due to interactions of the heme with different amino acid side chains in the near heme environment. MATERIALS AND METHODS Horse, tuna, pigeon, dog, and Candida krusei cytochromes c were purchased from Sigma. Rhodospirillum rubrum cytochrome C2 was prepared according to Sponholtz et al. (16). Iso-i and iso-2 cytochromes c from bakers' yeast were prepared according to Sherman et al. (17), and spider monkey, human, cow, turtle, and horse cytochromes c were prepared from heart tissue by the procedure of Margoliash and Walasek (18) as modified by Brautigan et al. (19). Before spectra were taken all preparations were filtered through a column of Sephadex G-50 superfine (Pharmacia) to completely separate polymeric material, which caused a characteristic background fluorescence and a decrease in sensitivity of the difference spectra resulting from the increase in noise level. In general, the spectra were obtained with ferrocytochrome c (minimal dithionite reduced) at a concentration of about 0.2 mM, buffered in 10 or 25 mM Tris acetate, pH 7.8. With the yeast iso-i cytochrome, a trace of mercaptoethanol was added to prevent disulfide dimer formation at cysteine-102. Spectra were obtained in Pyrex or quartz cells at room temperature, taking from 2 to 10 hr for completion. At the end of each run there were no changes in the Raman spectra that could be attributed to the formation of ferricytochrome c. On several occasions optical spectra were taken at the end of the run to further demonstrate that no autooxidation had occurred. Gel filtration before and after a run confirmed that no polymeric material was present or had formed. Raman difference spectra were obtained by a modification of the technique reported by Kiefer (20, 21) and will be described in detail elsewhere. Briefly, samples were placed in a cylindrical cell with a partition along a diameter such that the Abbreviation: RDS, Raman difference spectroscopy.
§ To whom correspondence should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
t On leave from School of Physics, Georgia Institute of Teghnology,
Atlanta, GA 30332; permanent address; Sandia Laboratories, D5114, Albuquerque, NM 87185.
3865
3866
Biophysics: Shelnutt et al.
two samples were alternately illuminated by the argon ion laser at 5287 A as the cell was rotated. Thus both samples could be examined for each frequency setting of the monochromator. As the cell was rotated a synchronous signal was sent to gating and counting electronics to allow independent accumulation of the data from each sample and subsequent independent storage in a Texas Instruments 960B computer. The spectral slit width was 2 cm-' and the spectrometer was advanced in steps ranging from 0.1 to 0.5 cm-1. The spectra were scanned 10-25 times to signal average the data, and up to 4096 points could be collected for each of the two spectra. Under these experimental conditions the Raman lines of cytochromes c display their natural linewidths. To obtain a difference spectrum the intensity of a Raman line in one spectrum was adjusted to equal the intensity of that line in the other spectrum and the two spectra were subtracted digitally. The difference spectrum of two closely spaced and equally intense Lorentzian lines has the shape of a derivative of the line. For small frequency differences, Av, between the two Lorentzians, the frequency difference (20-22) between the maximum and the minimum in the difference spectrum is larger (r/v/). If r is the full width at half height of the Lorentzian lines of peak intensity, I, then the intensity (peakto-valley) in the difference spectrum, Io, is related to Av by Io = 2.6IoAv/r. By using this relationship for small frequency differences and calculating a calibration curve for larger differences, the size of the frequency shift, Av, may be obtained from the intensity in the difference spectrum, provided the intensity of the Raman line in the two materials being compared is balanced. This balance is achieved by multiplying the intensity of one spectrum by a constant (balance ratio) prior to calculating the difference spectrum of that particular line. With this RDS technique very small frequency differences may be detected, because errors due to thermal drift and irreproducibility of the monochromator are eliminated by obtaining data for both samples at each setting of the spectrometer. The spectral resolution and the spectrometer frequency stepping interval that we used in this study have been shown from model calculations to introduce no larger than a 6% error in the frequency differences.
Proc. Natl. Acad. Sci. USA 76 (1979)
Frequency,
cm-1
FIG. 1. At the top, Raman difference spectrum of tuna cytochrome c versus horse cytochrome c. For these data the spectrometer was advanced in 0.1 cm-' increments. At the bottom, Raman spectra of tuna and horse ferrocytochromes c. The 750 cm-' line of tuna cytochrome c is 0.1 cm-1 higher in frequency than that of the horse protein.
vidual vibrational modes independently and thereby determine whether frequency differences exist. This is particularly important for samples in which the relative intensity of the Raman lines differ for the two proteins compared. For example, the R. rubrum cytochrome C2 difference spectrum with a balance ratio of 0.85 shows clear differences in the 1127, 1310, and 1584
I. c 0
c
RESULTS Ferrocytochrome c has absorption bands in the red-yellow region, from which strong resonance Raman scattering takes place (11). There is an absorption band near 5500 A (a band), which is a zero vibrational level transition, and a broad vibrational side band (f3 band) centered at about 5200 A. Excitation into the a band results in general enhancement of all the symmetry-allowed in-plane porphyrin modes, whereas excitation in the 3 band preferentially enhances the vibrational modes responsible for that portion of the vibrational side band. The report examines data obtained with excitation in the : band at 5287 A. This excitation wavelength results in enhancement of vibrational modes in the 700-1600 cm-1 region. Spectra of the 750 cm-1 line in horse and tuna cytochromes c are shown in Fig. 1. Although the frequency difference in this line for the two proteins is only 0.1 cm-1, it can be reproducibly determined from the RDS spectrum. Fig. 2 gives the difference spectra between horse cytochrome c and that of pigeon, human, bakers' yeast iso-i, and the cytochrome C2 of R. rubrum. The balance factor, by which the horse cytochrome c spectrum was multiplied to obtain each difference spectrum, is indicated in the figure. These factors were selected so as to balance indi-
c
E.a
cr
412
750
Horse
825a
851127
800
1000
Frequency,
1310
1200
14
cm'
FIG. 2. Raman difference spectra between a series of cytochromes and the horse protein. For these data the spectrometer was advanced in 0.5 cm-' increments. The Raman spectrum of horse ferrocytochrome c is shown at the bottom. The scale factor for each difference spectrum is indicated on the left. The balance factor by which the horse cytochrome c data were multiplied is indicated in the label for each difference spectrum. (Inset) Difference spectrum of the R. rubrum protein at a balance ratio of 1.4, for which the frequency difference for the 750 cm-1 line becomes apparent. c
Biophysics: Shelnutt et al.
cm-1 lines, while with a ratio of 1.4 the difference in the 750 cm-1 line becomes evident (see Fig. 2). There are no observable differences (
