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The effect of pH and hydrogen-deuterium exchange on the heme pocket structure of cytochrome c probed by resonance Raman spectroscopy
Journal of ELSEVIER
Journalof Molecular
MOLECULAR STRUCTURE Structure
349 (1995)
125-128
The Effect of pH and Hydrogen-Deuterium Exchange on the Heme Structure of Cytochrome c Probed by Resonance Raman Spectroscopy
Pocket
S. Diipnera, P. Hildebrandta, G. E. Heibela, F. Vanheckea, and A. G. Maukb aMax-Planck-Institut
fti Strahlenchemie,
Postfach 101365, D-454 13 Mtilheim, F.R.G.
bDepartment of Biochemistry, University of British Columbia, Vancouver, Canada V6T 123
1. INTRODUCTION
The heme protein cytochrome c (Cyt) serves as an electron carrier in the respiratory chain of aerobic organism [ 11. Prior to the redox processes, electrostatically stabilized complexes are formed between Cyt and its partner protein cytochrome c oxidase (CcO). In such complexes protein-protein interactions induce structural changes in the heme pocket of Cyt [2]. Using resonance Raman (RR) spectroscopy which selectively probes the vibrational bands of the heme group, it was found that the bound Cyt is (partially) converted into a new conformational state (state II) which may facilitate the electron transfer to cytochrome c oxidase (CcO) [3,4]. However, based on the RR spectra of the Cyt-Cc0 complexes a detailed structural analysis of this state is difficult due to the additional contributions of the two heme groups of CcO. On the other hand, it has been suggested that the conformational changes in Cyt induced upon binding to Cc0 may be closely related to those which occur in the unbound Cyt at alkaline pH [2,5]. Thus, a detailed knowledge of the structure of the alkaline forms of Cyt is desirable and may contribute to the understanding of the electron transfer reaction between Cyt and CcO.
2. EXPERIMENTAL
Preparation and purification of yeast iso-l cytochrome c and its point mutants are described elsewhere ([5], and references therein). The RR spectra were measured with 413-mn excitation using a monochromatic detection system [4]. The spectra were analyzed by a specially designed band fitting program which includes the option of comprising individual bands within groups so that the measured spectra could be simulated by a combination of appropriately weighted component spectra.
3. RESULTS
AND DISCUSSION
The RR spectra of the oxidized iso-l Cyt were measured in the pH range between 7.0 and 12.0 (Figure 1). The bands in the region between 1450 and 1660 cm-’ are of particular interest since their frequencies are correlated with the spin and coordination state of the heme 0022-2860/95/$09.50 0 1995 Elsevier XSDI 0022-2860(95)08725-7
Science
B.V.
All rights
reserved
126
1503.6
1504.6
,
I
1510
1550
1590
1630
1510
Av/cm-1 Figure
1. RR spectra of oxidized
!
I
I
1550
1590
I
I
1630
Av /cm-l iso-l
Cyt at different
pH values, excited
at 413 nm.
iron (spin state marker bands) [6]. Hence, they are expected to respond to changes of the ligation pattern which are known to occur in this pH range [5]. Indeed, we note distinct frequency shifts and intensity changes of these bands upon increasing the pH. In particular, above pH 9.5, the bands shifts to higher frequencies by more than 2 cm-’ while below pH 9.5 the spectral changes are much smaller. Recently, Mauk and co-workers have demonstrated that above pH 8.0 the neutral form of Cyt W(NN is converted to two alkaline forms [Cyt(Al), Cyt(A2)] in which the original axial methionine ligand (Met-80) of the heme iron is replaced by Lys-79 and Lys-73, respectively ([5] and further unpublished results). Above pH 10.5 a conversion to further, yet unidentified species was noted. This implies that four or even more different conformational states may contribute to the measured RR spectra. Since these species exhibit quite similar band frequencies, the individual peaks in the spectra in Figure 1 represent overlaps of several closely spaced components. In order to analyze the structures of these species as well as the pH-dependent equilibria, it is necessary to disentangle the complex spectra. However, employing a conventional band fitting procedure wouId not lead to unique results. This is only possible if the spectral parameters (frequencies, half widths and relative intensities of the bands) of the individual species are known. For the neutral species of the wild-type and mutant proteins these parameters can readily be determined from the spectra measured at pH
127
7.0. In order to determine the spectral parameters of Cyt(A1) and Cyt(A2), we analyzed the RR spectra of those mutants, in which the lysine residues 73 and 79 were replaced by an alanine, so that in each case only one alkaline state was formed. The RR spectra of these variant proteins between pH 8.0 and 9.0 were analyzed by the band fitting program, taking into account the contributions of the neutral form whose (known) spectral parameters were kept constant. In this way the spectral parameters of both Cyt(A1) and Cyt(A2) were obtained. The changes in the RR spectra upon increasing the pH above 9.5 indicate that new species are formed. These spectra were analysed in the same way as described above. Figure 2C shows the RR spectrum in the v3 band region of the wild-type Cyt at pH 12.0. Adjacent to the dominant band at 1506.5 cm-’ there is a weaker component at 1500.8 cm”, both of them corresponding to the mode v3 of a six-coordinate low-spin (6cLS) configuration. The small band at 1489.5 cm-t may indicate an additional five-coordinate high-spin form [6]. The same spectra were obtained for the Ala-73 and Ala-79 mutants, implying that in the two 6cLS species [Cyt(Hl), Cyt(H2)] the lysine ligands of the alkaline form are replaced by other ligands at higher pH values. The mode v3 is of particular interest since this mode has been shown to sensitively reflect conformational changes of Cyt upon binding to Cc0 [4]. Figure 2B displays the spectrum of Cyt bound to CcO. Again, two g3 components are visible corresponding to two conformational states. While the major lowfrequency component is at the same position as v3 of the neutral form of Cyt (Figure 2A), the additional high frequency component which originates from the conformational state II agrees very well with that of Cyt(H1). Such an agreement is also found in other regions of the spectrum. Thus, it is concluded that Cyt(H1) exhibits a similar heme pocket structure as state II, i. e. the conformational state of Cyt formed in the complex with CcO. This finding
1484
1500
1516
Avkm-q Figure 2. RR spectra of oxidized iso-l Cyt dissolved at pH 7.0 (A) and pH 12.0 (C), and bound to Cc0 (B). The excitation line was 413 nm.
128
implies that the pK, of the underlying conformational changes which is about 10.5 in solution is drastically lowered upon binding to CcO. Presumably, these conformational changes are initiated by the neutralization of the positive charges of the lysine residues in the binding domain (in particular Lys-72 and Lys-79) either via the deprotonation of the s-amino groups in solution [Cyt(Hl)] or via the formation of salt bridges with negatively charged amino acid side chains in the complex with Cc0 (state II). With respect to the biological function of Cyt, the structural similarity between Cyt(H1) and state II implies that the electron transfer from Cyt to Cc0 in the protein complex might be initiated by an exchange of the native axial ligand. Hence, we attempted to identify the nature of the sixth ligand in Cyt(H1). Coordination by the deprotonated Tyr-67, which is located close to Met-80 [7], can be ruled out since the RR spectra of the Phe-67 mutant at high pH values are essentially the same as those of the wild-type protein. Thus, the most likely candidate is a hydroxide ion, originating from the nearby water molecule WAT-166. The Fe-OH stretching vibration is expected in the region between 400 and 600 cm-’ which we have carefully examined using H,O and D,O solutions. In fact, isotopic shifts were noted for several bands. However, these effects were comparable to those observed for the neutral form of Cyt, so that none of these bands could be assigned to the Fe-OH stretching. Presumably, the Soret-band excitation used for these experiments does not provide a sufficient resonance enhancement for this mode. The observed isotopic shifts in the low frequency region are attributed to subtle conformational changes of the heme, due to the alteration of the hydrogen bonding network upon replacement of the exchangeable protons by deuterons [8]. We have determined the time-dependent evolution of these spectral changes, following rapid mixing of a concentrated Cyt solution with D,O buffer at pD 7.0. In this way a rate constant of 0.004 s-l was obtained which is significantly faster than the proton exchange reactions of the amide backbone [9]. This implies that the changes in the RR spectrum of the neutral form must be associated with the rapid proton exchange of the amino acid side chains and water molecules in the interior of the heme pocket. These findings strongly support a previous suggestion that the hydrogen bonding network in the heme pocket sensitively controls the porphyrin structure [S]. On the other hand, further experiments are required to identify the sixth ligand in both Cyt(H1) and Cyt(H2).
REFERENCES 1. G. W. Pettigrew and G. R. Moore, Cytochrome c - Biological Aspects, Springer, Berlin, 1987. 2. C. Weber, B. Michel, and H. R. Bosshard, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 6687. 3. P. Hildebrandt, T. Heimburg, D. Marsh, and G. L. Powell, Biochemistry, 29 (1990) 1661. 4. P. Hildebrandt, F. Vanhecke, G. Buse, T. Soulimane, and A. G. Mauk, Biochemistry, 32 (1993) 10912. 5. J. C. Ferrer, J. G. Guillemette, R. Bogumil, S. C. Iglis, M. Smith, and A. G. Mauk, J. Am. Chem. Sot., 115 (1993) 7507. 6. N. Parthasarathi, C. Hansen, S. Yamaguchi, and T. G. Spiro, J. Am. Chem. Sot., 109 (1987) 3865. 7. A. M. Berghuis and G. D. Brayer, J. Mol. Biol. 223 (1992) 959. 8. P. Hildebrandt, F. Vanhecke, G. Heibel, and A. G. Mauk, Biochemistry, 32 (1993) 14158. 9. L. Mayne, Y. Paterson, D. Cesaroli, and S. W. Englander, Biochemistry, 3 1 (1992) 10678.
Journalof Molecular
MOLECULAR STRUCTURE Structure
349 (1995)
125-128
The Effect of pH and Hydrogen-Deuterium Exchange on the Heme Structure of Cytochrome c Probed by Resonance Raman Spectroscopy
S. Diipnera, P. Hildebrandta, G. E. Heibela, F. Vanheckea, and A. G. Maukb aMax-Planck-Institut
fti Strahlenchemie,
Postfach 101365, D-454 13 Mtilheim, F.R.G.
bDepartment of Biochemistry, University of British Columbia, Vancouver, Canada V6T 123
1. INTRODUCTION
The heme protein cytochrome c (Cyt) serves as an electron carrier in the respiratory chain of aerobic organism [ 11. Prior to the redox processes, electrostatically stabilized complexes are formed between Cyt and its partner protein cytochrome c oxidase (CcO). In such complexes protein-protein interactions induce structural changes in the heme pocket of Cyt [2]. Using resonance Raman (RR) spectroscopy which selectively probes the vibrational bands of the heme group, it was found that the bound Cyt is (partially) converted into a new conformational state (state II) which may facilitate the electron transfer to cytochrome c oxidase (CcO) [3,4]. However, based on the RR spectra of the Cyt-Cc0 complexes a detailed structural analysis of this state is difficult due to the additional contributions of the two heme groups of CcO. On the other hand, it has been suggested that the conformational changes in Cyt induced upon binding to Cc0 may be closely related to those which occur in the unbound Cyt at alkaline pH [2,5]. Thus, a detailed knowledge of the structure of the alkaline forms of Cyt is desirable and may contribute to the understanding of the electron transfer reaction between Cyt and CcO.
2. EXPERIMENTAL
Preparation and purification of yeast iso-l cytochrome c and its point mutants are described elsewhere ([5], and references therein). The RR spectra were measured with 413-mn excitation using a monochromatic detection system [4]. The spectra were analyzed by a specially designed band fitting program which includes the option of comprising individual bands within groups so that the measured spectra could be simulated by a combination of appropriately weighted component spectra.
3. RESULTS
AND DISCUSSION
The RR spectra of the oxidized iso-l Cyt were measured in the pH range between 7.0 and 12.0 (Figure 1). The bands in the region between 1450 and 1660 cm-’ are of particular interest since their frequencies are correlated with the spin and coordination state of the heme 0022-2860/95/$09.50 0 1995 Elsevier XSDI 0022-2860(95)08725-7
Science
B.V.
All rights
reserved
126
1503.6
1504.6
,
I
1510
1550
1590
1630
1510
Av/cm-1 Figure
1. RR spectra of oxidized
!
I
I
1550
1590
I
I
1630
Av /cm-l iso-l
Cyt at different
pH values, excited
at 413 nm.
iron (spin state marker bands) [6]. Hence, they are expected to respond to changes of the ligation pattern which are known to occur in this pH range [5]. Indeed, we note distinct frequency shifts and intensity changes of these bands upon increasing the pH. In particular, above pH 9.5, the bands shifts to higher frequencies by more than 2 cm-’ while below pH 9.5 the spectral changes are much smaller. Recently, Mauk and co-workers have demonstrated that above pH 8.0 the neutral form of Cyt W(NN is converted to two alkaline forms [Cyt(Al), Cyt(A2)] in which the original axial methionine ligand (Met-80) of the heme iron is replaced by Lys-79 and Lys-73, respectively ([5] and further unpublished results). Above pH 10.5 a conversion to further, yet unidentified species was noted. This implies that four or even more different conformational states may contribute to the measured RR spectra. Since these species exhibit quite similar band frequencies, the individual peaks in the spectra in Figure 1 represent overlaps of several closely spaced components. In order to analyze the structures of these species as well as the pH-dependent equilibria, it is necessary to disentangle the complex spectra. However, employing a conventional band fitting procedure wouId not lead to unique results. This is only possible if the spectral parameters (frequencies, half widths and relative intensities of the bands) of the individual species are known. For the neutral species of the wild-type and mutant proteins these parameters can readily be determined from the spectra measured at pH
127
7.0. In order to determine the spectral parameters of Cyt(A1) and Cyt(A2), we analyzed the RR spectra of those mutants, in which the lysine residues 73 and 79 were replaced by an alanine, so that in each case only one alkaline state was formed. The RR spectra of these variant proteins between pH 8.0 and 9.0 were analyzed by the band fitting program, taking into account the contributions of the neutral form whose (known) spectral parameters were kept constant. In this way the spectral parameters of both Cyt(A1) and Cyt(A2) were obtained. The changes in the RR spectra upon increasing the pH above 9.5 indicate that new species are formed. These spectra were analysed in the same way as described above. Figure 2C shows the RR spectrum in the v3 band region of the wild-type Cyt at pH 12.0. Adjacent to the dominant band at 1506.5 cm-’ there is a weaker component at 1500.8 cm”, both of them corresponding to the mode v3 of a six-coordinate low-spin (6cLS) configuration. The small band at 1489.5 cm-t may indicate an additional five-coordinate high-spin form [6]. The same spectra were obtained for the Ala-73 and Ala-79 mutants, implying that in the two 6cLS species [Cyt(Hl), Cyt(H2)] the lysine ligands of the alkaline form are replaced by other ligands at higher pH values. The mode v3 is of particular interest since this mode has been shown to sensitively reflect conformational changes of Cyt upon binding to Cc0 [4]. Figure 2B displays the spectrum of Cyt bound to CcO. Again, two g3 components are visible corresponding to two conformational states. While the major lowfrequency component is at the same position as v3 of the neutral form of Cyt (Figure 2A), the additional high frequency component which originates from the conformational state II agrees very well with that of Cyt(H1). Such an agreement is also found in other regions of the spectrum. Thus, it is concluded that Cyt(H1) exhibits a similar heme pocket structure as state II, i. e. the conformational state of Cyt formed in the complex with CcO. This finding
1484
1500
1516
Avkm-q Figure 2. RR spectra of oxidized iso-l Cyt dissolved at pH 7.0 (A) and pH 12.0 (C), and bound to Cc0 (B). The excitation line was 413 nm.
128
implies that the pK, of the underlying conformational changes which is about 10.5 in solution is drastically lowered upon binding to CcO. Presumably, these conformational changes are initiated by the neutralization of the positive charges of the lysine residues in the binding domain (in particular Lys-72 and Lys-79) either via the deprotonation of the s-amino groups in solution [Cyt(Hl)] or via the formation of salt bridges with negatively charged amino acid side chains in the complex with Cc0 (state II). With respect to the biological function of Cyt, the structural similarity between Cyt(H1) and state II implies that the electron transfer from Cyt to Cc0 in the protein complex might be initiated by an exchange of the native axial ligand. Hence, we attempted to identify the nature of the sixth ligand in Cyt(H1). Coordination by the deprotonated Tyr-67, which is located close to Met-80 [7], can be ruled out since the RR spectra of the Phe-67 mutant at high pH values are essentially the same as those of the wild-type protein. Thus, the most likely candidate is a hydroxide ion, originating from the nearby water molecule WAT-166. The Fe-OH stretching vibration is expected in the region between 400 and 600 cm-’ which we have carefully examined using H,O and D,O solutions. In fact, isotopic shifts were noted for several bands. However, these effects were comparable to those observed for the neutral form of Cyt, so that none of these bands could be assigned to the Fe-OH stretching. Presumably, the Soret-band excitation used for these experiments does not provide a sufficient resonance enhancement for this mode. The observed isotopic shifts in the low frequency region are attributed to subtle conformational changes of the heme, due to the alteration of the hydrogen bonding network upon replacement of the exchangeable protons by deuterons [8]. We have determined the time-dependent evolution of these spectral changes, following rapid mixing of a concentrated Cyt solution with D,O buffer at pD 7.0. In this way a rate constant of 0.004 s-l was obtained which is significantly faster than the proton exchange reactions of the amide backbone [9]. This implies that the changes in the RR spectrum of the neutral form must be associated with the rapid proton exchange of the amino acid side chains and water molecules in the interior of the heme pocket. These findings strongly support a previous suggestion that the hydrogen bonding network in the heme pocket sensitively controls the porphyrin structure [S]. On the other hand, further experiments are required to identify the sixth ligand in both Cyt(H1) and Cyt(H2).
REFERENCES 1. G. W. Pettigrew and G. R. Moore, Cytochrome c - Biological Aspects, Springer, Berlin, 1987. 2. C. Weber, B. Michel, and H. R. Bosshard, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 6687. 3. P. Hildebrandt, T. Heimburg, D. Marsh, and G. L. Powell, Biochemistry, 29 (1990) 1661. 4. P. Hildebrandt, F. Vanhecke, G. Buse, T. Soulimane, and A. G. Mauk, Biochemistry, 32 (1993) 10912. 5. J. C. Ferrer, J. G. Guillemette, R. Bogumil, S. C. Iglis, M. Smith, and A. G. Mauk, J. Am. Chem. Sot., 115 (1993) 7507. 6. N. Parthasarathi, C. Hansen, S. Yamaguchi, and T. G. Spiro, J. Am. Chem. Sot., 109 (1987) 3865. 7. A. M. Berghuis and G. D. Brayer, J. Mol. Biol. 223 (1992) 959. 8. P. Hildebrandt, F. Vanhecke, G. Heibel, and A. G. Mauk, Biochemistry, 32 (1993) 14158. 9. L. Mayne, Y. Paterson, D. Cesaroli, and S. W. Englander, Biochemistry, 3 1 (1992) 10678.