/ . Biochem., 78, 719-728 (1975)
Effects of Ligands upon the Raman Spectra of Various C-type Cytochromes Teizo KITAGAWA,* Yoshimasa KYOGOKU,* Tetsutaro IIZUKA,** Masao IKEDA-SAITO,** and Tateo YAMANAKA*** "Institute for Protein Research, Osaka University, Yamadakami, Suita, Osaka 565, **Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560, and ***Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560 Received for publication, April 28, 1975
Resonance Raman spectra were measured for various C-type cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome /, and alkylated cytochrome c) and a B-type cytochrome (cytochrome b$) in their reduced and oxidized states. (1) For ferrous alkylated cytochrome c, a Raman line sensitive to the replacement of an axial ligand of the heme iron was found around 1540 cm"1. This ligand-sensitive Raman line indicated the transition from acidic (1545 cm"1) to alkaline (1533 cm"1) forms with pK 7.9. The pH dependence of the Raman spectrum corresponded well to that of the optical absorption spectra. (2) For ferrous cytochrome / , the ligand-sensitive Raman line was found at the same frequency as cytochrome c (1545 cm"1). Accordingly two axial ligands are likely to be histidine and methionine as in cytochrome c. (3) For ferrous cytochrome c 3 , the frequency of the ligand-sensitive Raman line was between those of cytochrome c and cytochrome bj. Since two axial ligands of the heme iron in cytochrome bb are histidines, most of the axial ligands of the four hemes in cytochrome C3 might be histidines. However, a combination of histidine and methionine as a possible set of two axial ligands was not completely excluded for one or two of the four hemes. (4) In ferrous cytochrome b5, two weak Raman lines appeared at 1302 and 1338 cm"1 instead of the strongest band at 1313 cm" 1 of C-type ferrous cytochromes. This suggests the practical use of these bands for the identification of types of cytochromes. The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical distortion of heme.
T
Abbreviations: alkylated cytochrome c, di-carboxymethyl-methionyl-cytochrome c; Hb, hemoglobin; Mb, myoglobin. Vol. 78, No. 4, 1975
719
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Resonance Raman Scattering from Hemoproteins
720
T. KITAGAWA, Y. KYOGOKU, T. IIZUKA, M. IKEDA-SAITO, and T. YAMANAKA
cytochrome c, but the redox potential is much lower (Emj= —0.28 V) as compared with mammalian cytochrome c. McDonald et al. (21) and Dobson et al. (22) have claimed that all eight ligands of four hemes of cytochrome c3 are histidines on the basis of its NMR spectra. Raman scattering can be used to check the NMR results. Cytochrome 65 isolated from rabit liver (23) is a typical low spin protoheme protein (24) in which two histidines are coordinated to heme iron (25). The Raman spectrum of cytochrome bs may be helpful in analysing the Raman spectrum of cytochrome c3 and should also reveal the difference in vibrational spectra between the two types of hemes. EXPERIMENTAL PROCEDURE Materials—The purification of horse cytochrome c and its alkylation were described elsewhere (26). Desulfovibrio vulgaris cytochrome c3 was highly purified by the method of Yagi and Maruyama (20). Cytochrome / was isolated from blue-green algae Spirulina platensis. The purification procedures will be described elsewhere (Yamanaka, T., unpublished results). Cytochrome b5, purified by the method of Omura and Takesue (23), was generously provided by Drs. K. Enomoto and R. Sato (Institute for Protein Research, Osaka University). Azide and imidazole complexes of horse cytochrome c were obtained by adding NaN3 and imidazole, respectively to a solution of ferric cytochrome c at pH 7.0. The pH values in acidic and alkaline regions were adjusted with HC1 and NaOH, respectively. Method—Measurements of Raman spectra were carried out using the 514.5 nm excitation line of an Ar + /Kr + mixed gas laser (Spectra Physics model 164-02) and a JEOL-02AS Raman spectrometer equipped with a cooled HTVR374 photomultiplier and photon counting detection. Calibration of the spectrometer was performed with indene (27) and D2 gas (28). The estimated errors of frequency and depolarization ratio are as large as 2 cm"1 and 0.1, respectively. For measurements of the Raman spectra of ferrous cytochromes, 200 pi of 0.1 mM cytoJ. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/78/4/719/779322 by Leiden University / LUMC user on 17 January 2019
Resonance Raman studies on hemoproteins have led to important discussions on the intensity enhancement mechanism of resonance Raman scattering (1—3), the effective symmetry of heme (4—6), and the binding nature of the 6th ligand with heme iron (7—13). From the similarity of HbO2 to ferric low spin derivatives of Hb in Raman spectra, Yamamoto et al. (10) pointed out that the iron in HbO2 is in the ferric state, as postulated in the Weiss model (14), while we suggested that the Raman spectra of ferrous low spin complexes may be used to distinguish two types of iron-ligand bonds, that is, a bonds and it bonds ( 15). Accordingly, in this report, we focused our attention on the effect of ligands upon the Raman spectra of various cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome / , alkylated cytochrome c, and cytochrome bs). In alkylated cytochrome c which has been carboxymethylated in the presence of KCN, lysine-79 is supposed to be coordinated to heme iron instead of methionine-80 (16). A pH-dependent change of the absorption spectrum of ferrous alkylated cytochrome c (17) was elucidated as a structural change associated with the change of coordination state of lysine-79 (18) and accordingly the pH dependence of the Raman spectra was examined carefully in the present study. Cytochrome / is a C-type cytochrome, the 5th and 6th ligands of which were unknown. Its spectral properties differ a little from those of mammalian cytochrome c(19), i.e. the ratio of absorbance of the Soret band to the a band, Ar/Aa is relatively high (~7) in comparison with cytochrome c (~4.5) and the a absorption band is asymmetric. Moreover, cytochrome / is generally an acidic protein while cytochrome c is basic and the midpoint redox potential is relatively high (Em,i = 0.34 V). These properties are supposed to depend upon two ligands of heme iron as well as the conformation of proteins just around heme. Resonance Raman spectra can give information on the ligation in cytochrome / . Cytochrome Cz from the bacterium Desulfovibrio vulgaris (20) is similar in molecular weight and absorption spectrum to mammalian
•4
RESONANCE RAMAN SCATTERING FROM HEMOPROTEINS
cyt-cOi;
cyt-
RESULTS Figure 1 illustrates the resonance Raman spectra of ferrous (II) and ferric (III) cytochromes in the frequency region above 1000 cm"1. In spite of the different apoproteins, the general patterns of Raman spectra of C-type cytochromes are closely similar. The strong band, seen in all C-type ferrous cytochromes, is missing in cytochrome b5 and two bands appear at 1302 and 1338 cm"1 instead. This difference may permit the practical use of Raman spectra to distinguish the two types of cytochromes. Raman spectra of ferrous cytochromes in the lower frequency region are shown in Fig. 2. A Raman spectral difference between B-type and C-type hemes is also seen around 970 cm"1 and 790-670 cm"1. The strong peak at 748 cm"1 of C-type cytochromes is the only depolarized band in this frequency region. Raman lines of ferric species were too weak to be observed in the lower frequency region. Figure 3 illustrates the polarized Raman spectra of ferrous and ferric alkylated cytochromes c at pH 9.5. Solid lines and broken lines denote that the electric vector of the scattered radiation is parallel (fy) and perpendicular (Ij_) to that of the incident laser light, Vol. 78, No. 4, 1975
Fig. 1. Raman spectra of various cytochromes. II and III denote ferrous and ferric states, respectively, cyt-c, cytochrome c; cyt-c3, cytochrome c3; cyt-f, cytochrome / ; alkyl-c, alkylated cytochrome c; and cyt-65, cytochrome b$. Instrumental conditions: excitation, 514.5 nm, power 200 mW, sensitivity 2000 counts/sec, rate 0.25 sec, time constant 0.5 sec, slit width 6 cm"1 for ferrous state, power 200 mW, sensitivity 1000 counts/sec, rate 0.25 sec, time constant 0.5 sec, slit width 7 cm"1, scan speed 6 A/min (20 cm"'/min) for ferric state.
respectively. On the basis of the polarization properties, Raman lines for Fe 2+ are related to
Downloaded from https://academic.oup.com/jb/article-abstract/78/4/719/779322 by Leiden University / LUMC user on 17 January 2019
chrome in 0.1 M phosphate buffer was contained in a cylindrical cell. To avoid auto-oxidation, we adopted the following method. After the protein solution had been degassed completely, a small amount of sodium dithionite was put on the frozen solution in the cell. Before the frozen solution melted, the cell was evacuated to 0.05 mmHg and the vacuum was maintained during the measurement to avoid oxidation. This technique was indispensable for measurement of the Raman spectra of ferrous cytochrome c3 and low pH solutions of other cytochromes. To obtain the Raman spectra of ferric cytochromes, potassium ferricyanide was added to 0.7 mM solutions of cytochromes. The lower front edge of the cell was irradiated with the excitation light and the temperature of the sample was kept around 10° by flushing with cold nitrogen gas. Absorption spectra were measured with a Hitachi 124 recording spectrophotometer at 25°.
721
722
T. KITAGAWA, Y. KYOGOKU, T. IIZUKA, M. IKEDA-SAITO, and T. YAMANAKA
those for Fe 3+ as shown in Table I. Except for the band around 1170 cm"1, Fe 3+ gives rise to Raman lines at higher frequency than Fe 2+ . Figure 4 shows the pH dependence of the
cyl-f(ll)
TABLE I. Comparison of frequencies between Fe2+ and Fe3+ alkylated cytochromes c (pH 9.5) [cm"1]. cyi-cClV
cyt-bgOl)
Fe2+ 1124 (ip)a 1172 (dp) 1228 (dp) 1240 (dp) 1311 (ap) 1358 (P) 1396 (ip) 1401 (dp) 1489 (P) 1541 (dp)
§
Fig. 2. Raman spectra of ferrous cytochromes in the low frequency region. Broad background below 500 cm"1 is due to the cell window, dp denotes a depolarized band; others are polarized. Instrumental conditions: power 250 mW, sensitivity 1000 counts/ sec, rate 0.25 sec, time constant 0.5 sec, slit width 7 cm"1, scan speed 2.4 A/min.
1583 (ip) 1620 (dp)
Fe 3 +
1169 (dp)
-3
1317 (ap) 1374 (p)
+6 + 16
1405 (ip) 1409 (dp) 1501 (p) 1564 (dp) 1587 (ip) 1636 (dp)
+9 +8 + 12 + 23
+4 + 16
a
Polarization properties are represented in accordance with the observed linear depolarization ratio (Pi = l±JIll)- P: polarized (Pi
Effects of Ligands upon the Raman Spectra of Various C-type Cytochromes Teizo KITAGAWA,* Yoshimasa KYOGOKU,* Tetsutaro IIZUKA,** Masao IKEDA-SAITO,** and Tateo YAMANAKA*** "Institute for Protein Research, Osaka University, Yamadakami, Suita, Osaka 565, **Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560, and ***Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560 Received for publication, April 28, 1975
Resonance Raman spectra were measured for various C-type cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome /, and alkylated cytochrome c) and a B-type cytochrome (cytochrome b$) in their reduced and oxidized states. (1) For ferrous alkylated cytochrome c, a Raman line sensitive to the replacement of an axial ligand of the heme iron was found around 1540 cm"1. This ligand-sensitive Raman line indicated the transition from acidic (1545 cm"1) to alkaline (1533 cm"1) forms with pK 7.9. The pH dependence of the Raman spectrum corresponded well to that of the optical absorption spectra. (2) For ferrous cytochrome / , the ligand-sensitive Raman line was found at the same frequency as cytochrome c (1545 cm"1). Accordingly two axial ligands are likely to be histidine and methionine as in cytochrome c. (3) For ferrous cytochrome c 3 , the frequency of the ligand-sensitive Raman line was between those of cytochrome c and cytochrome bj. Since two axial ligands of the heme iron in cytochrome bb are histidines, most of the axial ligands of the four hemes in cytochrome C3 might be histidines. However, a combination of histidine and methionine as a possible set of two axial ligands was not completely excluded for one or two of the four hemes. (4) In ferrous cytochrome b5, two weak Raman lines appeared at 1302 and 1338 cm"1 instead of the strongest band at 1313 cm" 1 of C-type ferrous cytochromes. This suggests the practical use of these bands for the identification of types of cytochromes. The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical distortion of heme.
T
Abbreviations: alkylated cytochrome c, di-carboxymethyl-methionyl-cytochrome c; Hb, hemoglobin; Mb, myoglobin. Vol. 78, No. 4, 1975
719
Downloaded from https://academic.oup.com/jb/article-abstract/78/4/719/779322 by Leiden University / LUMC user on 17 January 2019
Resonance Raman Scattering from Hemoproteins
720
T. KITAGAWA, Y. KYOGOKU, T. IIZUKA, M. IKEDA-SAITO, and T. YAMANAKA
cytochrome c, but the redox potential is much lower (Emj= —0.28 V) as compared with mammalian cytochrome c. McDonald et al. (21) and Dobson et al. (22) have claimed that all eight ligands of four hemes of cytochrome c3 are histidines on the basis of its NMR spectra. Raman scattering can be used to check the NMR results. Cytochrome 65 isolated from rabit liver (23) is a typical low spin protoheme protein (24) in which two histidines are coordinated to heme iron (25). The Raman spectrum of cytochrome bs may be helpful in analysing the Raman spectrum of cytochrome c3 and should also reveal the difference in vibrational spectra between the two types of hemes. EXPERIMENTAL PROCEDURE Materials—The purification of horse cytochrome c and its alkylation were described elsewhere (26). Desulfovibrio vulgaris cytochrome c3 was highly purified by the method of Yagi and Maruyama (20). Cytochrome / was isolated from blue-green algae Spirulina platensis. The purification procedures will be described elsewhere (Yamanaka, T., unpublished results). Cytochrome b5, purified by the method of Omura and Takesue (23), was generously provided by Drs. K. Enomoto and R. Sato (Institute for Protein Research, Osaka University). Azide and imidazole complexes of horse cytochrome c were obtained by adding NaN3 and imidazole, respectively to a solution of ferric cytochrome c at pH 7.0. The pH values in acidic and alkaline regions were adjusted with HC1 and NaOH, respectively. Method—Measurements of Raman spectra were carried out using the 514.5 nm excitation line of an Ar + /Kr + mixed gas laser (Spectra Physics model 164-02) and a JEOL-02AS Raman spectrometer equipped with a cooled HTVR374 photomultiplier and photon counting detection. Calibration of the spectrometer was performed with indene (27) and D2 gas (28). The estimated errors of frequency and depolarization ratio are as large as 2 cm"1 and 0.1, respectively. For measurements of the Raman spectra of ferrous cytochromes, 200 pi of 0.1 mM cytoJ. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/78/4/719/779322 by Leiden University / LUMC user on 17 January 2019
Resonance Raman studies on hemoproteins have led to important discussions on the intensity enhancement mechanism of resonance Raman scattering (1—3), the effective symmetry of heme (4—6), and the binding nature of the 6th ligand with heme iron (7—13). From the similarity of HbO2 to ferric low spin derivatives of Hb in Raman spectra, Yamamoto et al. (10) pointed out that the iron in HbO2 is in the ferric state, as postulated in the Weiss model (14), while we suggested that the Raman spectra of ferrous low spin complexes may be used to distinguish two types of iron-ligand bonds, that is, a bonds and it bonds ( 15). Accordingly, in this report, we focused our attention on the effect of ligands upon the Raman spectra of various cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome / , alkylated cytochrome c, and cytochrome bs). In alkylated cytochrome c which has been carboxymethylated in the presence of KCN, lysine-79 is supposed to be coordinated to heme iron instead of methionine-80 (16). A pH-dependent change of the absorption spectrum of ferrous alkylated cytochrome c (17) was elucidated as a structural change associated with the change of coordination state of lysine-79 (18) and accordingly the pH dependence of the Raman spectra was examined carefully in the present study. Cytochrome / is a C-type cytochrome, the 5th and 6th ligands of which were unknown. Its spectral properties differ a little from those of mammalian cytochrome c(19), i.e. the ratio of absorbance of the Soret band to the a band, Ar/Aa is relatively high (~7) in comparison with cytochrome c (~4.5) and the a absorption band is asymmetric. Moreover, cytochrome / is generally an acidic protein while cytochrome c is basic and the midpoint redox potential is relatively high (Em,i = 0.34 V). These properties are supposed to depend upon two ligands of heme iron as well as the conformation of proteins just around heme. Resonance Raman spectra can give information on the ligation in cytochrome / . Cytochrome Cz from the bacterium Desulfovibrio vulgaris (20) is similar in molecular weight and absorption spectrum to mammalian
•4
RESONANCE RAMAN SCATTERING FROM HEMOPROTEINS
cyt-cOi;
cyt-
RESULTS Figure 1 illustrates the resonance Raman spectra of ferrous (II) and ferric (III) cytochromes in the frequency region above 1000 cm"1. In spite of the different apoproteins, the general patterns of Raman spectra of C-type cytochromes are closely similar. The strong band, seen in all C-type ferrous cytochromes, is missing in cytochrome b5 and two bands appear at 1302 and 1338 cm"1 instead. This difference may permit the practical use of Raman spectra to distinguish the two types of cytochromes. Raman spectra of ferrous cytochromes in the lower frequency region are shown in Fig. 2. A Raman spectral difference between B-type and C-type hemes is also seen around 970 cm"1 and 790-670 cm"1. The strong peak at 748 cm"1 of C-type cytochromes is the only depolarized band in this frequency region. Raman lines of ferric species were too weak to be observed in the lower frequency region. Figure 3 illustrates the polarized Raman spectra of ferrous and ferric alkylated cytochromes c at pH 9.5. Solid lines and broken lines denote that the electric vector of the scattered radiation is parallel (fy) and perpendicular (Ij_) to that of the incident laser light, Vol. 78, No. 4, 1975
Fig. 1. Raman spectra of various cytochromes. II and III denote ferrous and ferric states, respectively, cyt-c, cytochrome c; cyt-c3, cytochrome c3; cyt-f, cytochrome / ; alkyl-c, alkylated cytochrome c; and cyt-65, cytochrome b$. Instrumental conditions: excitation, 514.5 nm, power 200 mW, sensitivity 2000 counts/sec, rate 0.25 sec, time constant 0.5 sec, slit width 6 cm"1 for ferrous state, power 200 mW, sensitivity 1000 counts/sec, rate 0.25 sec, time constant 0.5 sec, slit width 7 cm"1, scan speed 6 A/min (20 cm"'/min) for ferric state.
respectively. On the basis of the polarization properties, Raman lines for Fe 2+ are related to
Downloaded from https://academic.oup.com/jb/article-abstract/78/4/719/779322 by Leiden University / LUMC user on 17 January 2019
chrome in 0.1 M phosphate buffer was contained in a cylindrical cell. To avoid auto-oxidation, we adopted the following method. After the protein solution had been degassed completely, a small amount of sodium dithionite was put on the frozen solution in the cell. Before the frozen solution melted, the cell was evacuated to 0.05 mmHg and the vacuum was maintained during the measurement to avoid oxidation. This technique was indispensable for measurement of the Raman spectra of ferrous cytochrome c3 and low pH solutions of other cytochromes. To obtain the Raman spectra of ferric cytochromes, potassium ferricyanide was added to 0.7 mM solutions of cytochromes. The lower front edge of the cell was irradiated with the excitation light and the temperature of the sample was kept around 10° by flushing with cold nitrogen gas. Absorption spectra were measured with a Hitachi 124 recording spectrophotometer at 25°.
721
722
T. KITAGAWA, Y. KYOGOKU, T. IIZUKA, M. IKEDA-SAITO, and T. YAMANAKA
those for Fe 3+ as shown in Table I. Except for the band around 1170 cm"1, Fe 3+ gives rise to Raman lines at higher frequency than Fe 2+ . Figure 4 shows the pH dependence of the
cyl-f(ll)
TABLE I. Comparison of frequencies between Fe2+ and Fe3+ alkylated cytochromes c (pH 9.5) [cm"1]. cyi-cClV
cyt-bgOl)
Fe2+ 1124 (ip)a 1172 (dp) 1228 (dp) 1240 (dp) 1311 (ap) 1358 (P) 1396 (ip) 1401 (dp) 1489 (P) 1541 (dp)
§
Fig. 2. Raman spectra of ferrous cytochromes in the low frequency region. Broad background below 500 cm"1 is due to the cell window, dp denotes a depolarized band; others are polarized. Instrumental conditions: power 250 mW, sensitivity 1000 counts/ sec, rate 0.25 sec, time constant 0.5 sec, slit width 7 cm"1, scan speed 2.4 A/min.
1583 (ip) 1620 (dp)
Fe 3 +
1169 (dp)
-3
1317 (ap) 1374 (p)
+6 + 16
1405 (ip) 1409 (dp) 1501 (p) 1564 (dp) 1587 (ip) 1636 (dp)
+9 +8 + 12 + 23
+4 + 16
a
Polarization properties are represented in accordance with the observed linear depolarization ratio (Pi = l±JIll)- P: polarized (Pi
