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EPR studies of protein state transitions in Chromatium ferricytochrome c ′
Biochimica et Biophysica Acta, 379 (1975)r95-102
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 36917 EPR STUDIES OF P R O T E I N STATE T R A N S I T I O N S IN C H R O M A T I U M F E R R I C Y T O C H R O M E c'
MARTIN M. MALTEMPO* Department of Physics, Columbia University, New York, N.Y. 10027 and IBM Thomas J. Watson Research Center, Yorktown Heights, N.Y. 10598 (U.S.A.)
(Received April 25th, 1974) (Revised manuscript received September 16th, 1974)
SUMMARY Recent magnetic studies have shown that in the pH range 1 to 11 the bacterial heme protein ferricytochrome c' can undergo reversible transitions between various pure high-spin and quantum mechanically mixed (intermediate and high)-spin protein states. The EPR data presented here extend the recent work to high alkaline p H and show that above pH 11.6 reversible transitions occur between various pure high-spin and pure low-spin protein states. The new data and the previous magnetic studies of the protein are discussed in the context of the entatic nature of the iron-porphyrin complex in ferricytochrome c'. EPR data are also provided concerning an anomalous, but reversible, transition to a protein state with reduced heme iron, triggered by freezing and thawing at physiological pH.
INTRODUCTION Recently, it has been shown that in the pH range 1 to 11 the heme protein, ferricytochrome c', isolated from the photosynthetic bacteria Cbromatium, can undergo reversible transitions between four different protein states, two high-spin states and two states corresponding to quantum mechanical admixtures of intermediate and high-spin states [1]. As such, ferricytochrome c' has been proposed as a particularly interesting example of a protein in an "entatic" or "poised" state [1 ]. The EPR studies reported here for Chromatium ferricytochrome c' support this view, indicating that above pH 11.6 the protein undergoes reversible transitions to additional high- and low-spin states. The magnetic properties and pH dependence of these states will be discussed in the context of previous magnetic studies at high alkaline p H [2], as well as the studies performed at physiological and acidic pH [1,2]. We will also discuss EPR results for ferricytochrome c' at pH 8.6 which indicate that a reduced iron complex can be stabilized by freezing and thawing of the protein. * Current address: Johnson Research Foundation, School of Medicine, University of Pennsylvania, Philadelphia, Pa. 19174, U.S.A.
96 METHODS
The isolation and purification of the Chromatium cytochrome c' used in this study has been described in previous publications [1,3]. All measurements at alkaline pH were made on protein in mixed-ion buffer 200 mM in each of glycine, sodium acetate, sodium phosphate and Tris. The protein used in the EPR studies at pH 8.6 was in a 25 mM buffer, with the same ion constituents as used in the alkaline measurements. Protein concentrations were determined from optical absorption measurements made using a Cary model 14 spectrophotometer, assuming a molar heme extinction coefficient of 84.4 mM -1.cm -1 for the 400-nm Soret band of the oxidized protein at pH 7.0. EPR measurements were made with a Varian E-9 X-band spectrometer. Protein concentrations were in the range 0.3 to 1.0 mM in heine iron. The protein samples were cooled to as low as 7 °K by liquid helium gas flow, as described elsewhere [1 ]. RESULTS
The high alkaline pH EPR measurements reported here for Chromatium ferricytochrome c' were made on four different protein samples, obtained from two different protein preparations. In each case the protein samples were first monitored by EPR in the pH range 7 to 11, where each sample showed the pH-dependent interconversion between protein states As, A2 and B2, characteristic of ferricytochrome c' [1]. The pH of the protein was then increased slowly above pH 11.0 by dialyzing the protein overnight against buffer at 4 °C. No change in the EPR spectrum of the protein was observed until the pH of the protein was increased to 11.7, where a sharp spectral transition was observed. Fig. 1 shows the EPR spectrum of one of the protein samples at pH 11.7 and 21 °K, before the transition has occurred, where the protein still has an EPR spectrum characteristic of the high-spin state B2*. The sample shown in Fig. 1 was then thawed,
pH 11,7AND 21°K, PRIOR TO HIGHALKALINE SPECTRAL TRANSITION
# I100
22'00 -
53'00
MAGNETIC FIELD (gouss)
Fig. 1. EPR spectrum of Chromatium ferricytochrome c' at pH ll.7 and 21 °K, prior to the high alkaline spectral transition. The microwave power was 10.0 roW; the frequency was 9.18 GHz. * The EPR spectra shown in this study contain an isotropic signal at about 1525 G, i.e. g .... 4.28. Resonances occurring at this value of magnetic field correspond to paramagnetic species in environments of purely rhombic symmetry and are quite common impurities in iron protein.
97 ~
'°tit
pH 11.7AND 15" K,
A TER T.E .,GH C3
I100
cC3_z B
2200 , MAGNETIC FIELD (gauss)
33~00
Fig. 2. EPR spectrum of Chromatium ferricytochrome c' at pH 11.7 and 15 °K, after the high alkaline spectral transition. The microwave power was 10.0 roW; the frequency was 9.18 GHz.
d i a l y z e d against buffer for 2 m o r e days at 4 °C a n d refrozen. T h e p H o f the s a m p l e was again m e a s u r e d to be a b o u t 11.7, b u t a new E P R s p e c t r u m was o b t a i n e d at 15 °K, as shown in Fig. 2. E a c h o f the other three p r o t e i n samples showed a similar high p H spectral transition. It a p p e a r s that the spectral t r a n s i t i o n is either slowly time d e p e n d e n t , o r it is specific to a quite n a r r o w p H range near 11.7. T h o u g h some o f the p a r a m a g n e t i c signals present in Fig. 2 are p a r t i a l l y s a t u r a t e d at 10 m W m i c r o w a v e power, extensive E P R m o n i t o r i n g o f ferricytoc h r o m e c' in the p H range 11.7 to 12.4 indicates that the t r a n s i t i o n at p H 11.7 results in five new E P R spectral states: B2' , B3, C1, C 2 a n d C3. The three spectral c o m p o n e n t s c o m m o n to each state were identified on the basis o f t e m p e r a t u r e - d e p e n d e n t b r o a d e n i n g , as well as p H a n d s a t u r a t i o n effects. These spectral c o m p o n e n t s are labeled in Fig. 2 a c c o r d i n g to the a p p r o p r i a t e E P R spectral state. The five h i g h - p H spectral states can be listed in o r d e r o f ease o f saturation as C1 > C2 > C3 ~ B2' ~ B3. The g values associated with each o f the spectral states are listed in T a b l e I, along with the g values o f the previously discussed p r o t e i n
TABLEI EPR g VALUES OF FERRICYTOCHROME c" PROTEIN STATES Protein state
g values a
pH b ( ~ amount)
A~ A2 Bt B2 B2"
4.75, 1.99 [1] 5.27, 1.99 [1] 5.94, 2.02 [1] 6.14,5.68,2.0011] 6.16, 5.73, 1.99 7.06, 4.73 ± 0.02 c 2.45, 2.07, 1.98 2.94, 2.23, 1.66 3.23, 2.31 c 2.022, 2.017, 2.004 d
7.0 (98%) [1] 10.0 ( ~ 33~) [1] 1.0 (> 99~) [i] 11.0(>99%)[1] 1t.7+ I 1.7 + 11.7-11.9 12.4 11.7+ 8.6
B3 C1 C2 C3 D
=g values are accurate to !0.01, unless otherwise indicated. h The pH at which there is the largest number of ferricytochrome c" molecules in a particular protein state (pH 11.7+ - pH where high alkaline spectral transition has occurred). c Existence of third EPR signal component uncertain. d The g values pertain to a hyperfine-split free radical signal.
98 states of ferricytochrome c':Ba (pH 1), A1 (pH 7), A2 (pH 10), and B2 (pH l l) [1], For an anisotropic signal monitored in the pH range 11.7-12.4, the g values of the EPR derivative signal were assigned to the low-field maximum, the high-field minimum, and the mid-field zero-crossing. Care was exercised to monitor the various EPR spectral species at values of pH and microwave power when neighbouring species were either absent or power saturated. Though the EPR signals of protein state B2 and spectral state B2' are similar, there are differences in the temperature-dependent line shape of the two EPR signals. In the present studies, as well as previous work [1 ], the rhombic splitting of the EPR signal of protein state B2 in the region of g -- 6 was clearly visible as two distinct peaks below 40 °K. However, the high-field component of the rhombically split signal of State B2' (in the region o f g = 6) is visible only as a shoulder, even in spectra obtained at 16 °K. Though Table I indicates that there is a small difference in the apparent g values associated with the two EPR signals, the significance of this measurement is uncertain due to the different temperature-dependent line shape of the two signals. After the sharp transition depicted in Fig. 2 has taken place, EPR spectra indicate that there are comparable amounts of protein in each of the high-pH EPR spectral states. As the pH of ferricytochrome c' is increased above 11.7, the intensity of signals B2', Ba and C3 decrease, until at pH 12.4 almost all of the protein ( ~ 95 ~o) has been converted to EPR spectral states Ca, and C2. Fig. 3 shows the EPR spectrum of ferricytochrome c' obtained at pH 12.4 and 31 °K, using 1.0 mW microwave power. The EPR data indicate that each of the five high-pH spectral states has a different, reversible pH titration curve. A close examination of Fig. 1 shows that a small, but measurable, amount of protein has been converted to EPR spectral state Ba, in the absence of any observable amount of protein in the other high-pH spectral states. A small B 3 EPR signal was also observed in spectra taken at pH l l.4, but not in spectra obtained at pH ~ 11.2. When the pH of ferricytochrome c' was decreased below l l.7, the protein changed reversibly back to protein state Bz and, if the pH was lowered further, to protein states A1, A2 and Ba. However, it was found that the backward transitions from the high-pH spectral states, C2, C1, C3, Bz' and Ba, down through protein states
C2
,
C3C2
pH 12.4AND 31° K
.60
2~oo
3~oo
MAGNETIC FIELD (gauss}
Fig. 3. EPR spectrum of Chromatium ferricytochrome c" at pH 12.4 and 31 °K, using 1.0 mW microwave power. The microwave frequency was 9.18 GHz.
99 B2, A1, A2 and B1, usually occurred at somewhat lower values of pH than the corresponding forward transitions. Protein samples in this study which showed this hysteresis property (after exposure to pH _>--11.7 for at least 24 h) were found to largely lose this property if the samples were "recycled" twice by titrations between pH 7 and p H 10. It should be briefly noted that in one of the samples used in this study, an anomalous, but reversible EPR spectral transition was observed upon freezing and thawing the sample at pH 8.6, prior to titration at high alkaline pH. The new spectral state, D, was characterized by an orange-red color (as compared with the normally light brown color of the protein at physiological pH), and an EPR spectrum in which there was a narrow 3-fold split paramagnetic signal in the region of g = 2, and a small paramagnetic iron signal in the region of g ---- 6 (reduced to less than 0.5 ~ of the signal observed prior to thawing). The characterization of the signal near g = 2, in spectral species D, was hindered by the presence of an overlapping background signal from a cavity contaminant. However, three sharp minima could be clearly observed at g = 2.002, 2.017 and 2.022, separated by about 16 G. Unfortunately, thawing this sample after the EPR run resulted in a complete spectral and pigment transition of the protein back to the "normal" protein states As, A2 and B2 [1], preventing any further characterization of spectral state D. In the succeeding pH titration from pH 7 to pH 12, this protein sample showed EPR spectra typical of other samples of ferricytochrome c'. There was no further observed change in color, and no appearance of the anomalous EPR signal in the region of g ----2. PROTEIN STATE TRANSITIONS pH~
! •o. PROTEIN STATE KEY A=QUANTUM MECHANICALLY MIXED INTERMEDIATE AND HIGH SPIN B = HIGH SPIN C= LOW SPIN D = REDUCED IRON
/J.~, TRANSITION KEY =pH DEPENDENT ......... FREEZING AND THAWING
Fig. 4. Protein state transitions in Chromatium ferricytochrome c'. The midpoint pH values of some of the transitions are indicated [1] (Maltempo, M. M., unpublished). The number of protein states monitored in the pH range 11.7-12.4, made a determination of midpoint pH values for these transitions difficult.
The observed EPR spectral transitions of Chromatium ferricytochrome c', including those previously reported [1] are shown schematically in Fig. 4. It should be noted that there was no indication in our magnetic studies that the two hemes in the Chromatium ferricytochrome c' dimer are in any way magnetically inequivalent.
100 DISCUSSION The data presented here indicate that above pH 11.7 Chromatium ferricytochrome c' undergoes reversible transitions between five magnetically distinguishable protein states, two high spin and three low spin. In addition, EPR data obtained at pH 8.6 indicate that a reduced iron complex can be stabilized in the oxidized protein by freezing and thawing. These matters will now be discussed in detail. Since each of the high alkaline pH spectral states has a different pH titration curve, we can conclude that each corresponds to a chemically distinct Chromatium ferricytochrome c' protein state. That is, in the temperature and pH range considered, the data presented here rules out the possibility of thermal mixtures of the various magnetic species, arising from ground and excited electronic configurations of the same heme-iron complex. A comparison of the g values and line shape of the various high alkaline pH EPR signals and typical high- and low-spin ferric heme EPR signals [4-6], indicates that protein states B 2' and B 3 are high-spin states and protein states Cj, C 2 and Ca are low-spin states. The symmetry at the iron site in protein states B 2' and B3 is essentially axial, as in other ferric high-spin heme-iron configurations, with the EPR g values indicating 15~o rhombicity [4] in state B 3 and 2 . 7 ~ in state B2'. The EPR g values indicate that the low-spin protein states can be listed in the order of increasing spatial anisotropy at the iron site as: Ca < C2 < Ca. It was pointed out in the preceeding section that while the EPR spectrum of protein state B E has a distinct rhombic splitting in the region of g ~ 6 below 40 °K, rhombic anisotropy in the EPR spectrum of state 12' is manifested only as a highfield shoulder on the g ~ 6 signal, even at 16 °K. The difference between the EPR signals of protein states B 2 and B 2' can be explained by assuming that after the titration of some protein group at pH 11.7, either the relaxation properties of ferricytochrome c' are altered, or more likely, steric strain at the iron site is somewhat "relaxed", resulting in an inhomogeneity in the rhombicity of the protein [7] and a less distinct rhombic splitting in the EPR spectrum. As discussed in the previous section, except for the presence of a small amount of protein in State B3 (Fig. 2), all of the molecules of ferricytochrome c' are in protein state B E until the sharp transition at pH 11.7; whereupon protein molecules are monitored in States B2', C1, C 2 and Ca, as well as in State Ba. However, it would seem quite unlikely that a number of sharp transitions from protein state B2 would all occur at about the same value of pH. Moreover, it was noted that during titratiom from physiological pH to high alkaline pH, the EPR spectral transitions to States C~, C2 and C3 were never observed before the appearance of signal I]2' , and that as the pH was increased above l l.7, the intensity of signals C1 and C2 increased at the expense of signal B2'. These data, taken in conjunction with the similarity in the magnetic properties of States 8 2 and B2', suggest that protein state B 2' may be a metastable intermediate bridging protein state B2 and the low-spin states. In this scheme, protein molecules can undergo a transition from protein state B2 to either the metastable protein state B 2' or protein state B3, but not directly to any of the low-spin states. However, as soon as the transition from Bz to B 2' has taken place, protein molecules can undergo transitions to the various low-spin states. This scheme is consistent with the EPR
101 data and avoids the alternate, unlikely explanation that there are a number of sharp transitions occurring with the same midpoint pH values. Other workers monitoring Chromatium and R. rubrum ferricytochrome c' by EPR, have previously reported the appearance of a new high-spin state at pH 11.6, followed by a transition to a low-spin state as the pH increased above 11.6, though no details were provided concerning the g values or line shape of the signals [2]. The transition to the low-spin state was reported to be complete at pH 12.1 in the R. rubrum protein, but still incomplete in the Chromatium protein at pH 12.0. The earlier study is in general agreement with our data; however, in the absence of detailed EPR data in the previous work it is unclear which two alkaline states were monitored. Our data indicate that at pH 12.4 almost all (=> 95 ~ ) of the protein has been converted to the low-spin states C~ and C2. Previous work, in which Chromatium ferricytochrome c' was monitored by optical absorption spectroscopy, has shown that the spectral transition from protein state B2 to the high alkaline pH spectral states is completely reversible when the pH is neutralized within a few minutes of the forward transition (Cusanovich, M. A., unpublished). In the context of the previous work, the observation of a pH hysteresis effect in our alkaline-treated protein samples may be a result of our method of pH titration, in which protein samples were dialyzed overnight against buffer at 4 °C, or simply the ability of EPR to provide more specific iron-heine magnetic data than optical absorption spectroscopy. Alkaline-treated protein samples which showed the hysteresis property were found to largely lose this property after being "recycled" twice by titrations between pH 7 and pH 10. EPR spectral State D, monitored in one of the protein samples at pH 8.6 prior to alkaline treatment, has not been observed in any of our previous studies [1]. The absence of a strong EP R iron signal suggests the possibility that protein aggregation has occurred, resulting in excessive dipole broadening of the paramagnetic iron signal. However, this possibility must be considered unlikely in view of the low protein concentration, the appearance of the g ~ 2 signal, and the complete reversibility of the transition to State D. It seems more likely that the iron ion has been reduced in State D, and that the protein state is a genuine, if somewhat anomalous, state of cytochrome c'. This view is supported by the EPR spectra of that sample in the pH range 7 to 12, which (except for the observed transition to State D) are typical of other sample spectra of ferricytochrome c'. The g values, line width and saturation properties of the g ~ 2 EPR signal indicate that it is probably a hyperfine-split free radical signal. The 3-fold splitting of the signal, and the apparent reduced state of the iron ion, would alone suggest the possibility that the transition to State D is accompanied by reversible charge transfer from, in all likelihood, an axial nitrogen ligand (I = 1), to the iron ion. However, this interpretation must be considered quite speculative, since a rough calculation indicates that the g ~ 2 signal in State D represents at least an order of magnitude less electron spin than that represented by the iron signal prior to the protein state transition. In summary, the data presented here, in conjunction with previous work [1], show that Chromatium ferricytochrome c' can undergo reversible transitions between ten magnetically distinguishable protein states. Nine protein states have oxidized heme iron: four (Ba, B2, B2' and Ba) are high spin, three (Ca, C2 and Ca) are low spin, and two (A~ and A2) are quantum mechanical admixtures of intermediate and
102 high-spin states [1]. The reversible transitions between these nine protein states are pH dependent. The remaining protein state, D, is somewhat anomalous, having been monitored in only one sample of ferricytochrome c'. EPR data for State D indicate that the iron ion is probably reduced, perhaps stabilized by reversible chargetransfer to the iron ion from an axial nitrogen ligand, triggered by freezing and thawing. It has been suggested that the quantum mechanically mixed-spin ferric heine complexes which predominated in ferricytochrome c' at physiological pH can be regarded magnetically (and perhaps structurally, as well) as "bridges" between the high- and low-spin ferric heme complexes [8]. The ability of ferricytochrome c' to undergo reversible transitions between these unique mixed-spin states and various high- and low-spin states provides new evidence for the entatic nature of the iron-porphyrin complex. It is hoped that future work will consider the role which the various protein states of ferricytochrome c' play in the biological function of the protein*, and whether there exists a correlation between the magnetic properties of the various states and the corresponding chemical properties, such as the redox potential. There is also the very interesting question of what role the ferricytochromes c' played in heme protein evolution. The iron-heme structural configuration inferred from the magnetic data at physiological pH [1] suggests the possibility that these unusual cytochromes may be evolutionary intermediates between high- and low-spin heme proteins. ACKNOWLEDGEMENTS The author wishes to thank Ms Edith R. Shapiro for her valuable technical assistance in the preparation and purification of Chromatium cytochrome c', and Dr Thomas H. Moss for innumerable, helpful discussions. REFERENCES 1 Maltempo, M. M., Moss, T. H. and Cusanovich, M. A. (1974) Biochim. Biophys. Acta 342, 290-305 2 Ehrenberg, A. and Kamen, M. D. (1965) Biochim. Biophys. Acta 102, 333-340 3 Cusanovich, M. A. (1967) Ph. D. Thesis, University of California at San Diego, U.S.A. 4 Peisach, J., Blumberg, W. E., Ogawa, S., Rachmilewitz, E. A. and Oltzik, R. (1971) J. Biol. Chem. 246, 3342-3355 5 Salmeen, I. and Palmer, G. (1968) J. Chem. Phys. 48, 2049-2052 6 Salmeen, I. and Palmer, G. (1968) J. Chem. Phys. 48, 4331"* 7 Mailer, C. and Taylor, C. P. S. (1972) Can. J. Biochem. 50, 1048-1055 8 Maltempo, M. M. (1974) J. Chem. Phys., 61, 2540-2547
* Though cytochrome c' is generallythought to be involvedin the cytochrome electron-transport system in bacteria, the specific function of the protein is unknown. The possibility that a protein state monitored in the isolated protein at acidic or alkaline pH might be biologically active in vivo should not be excluded, since it is conceivable that these states could be stabilized by conditions other than acidic or alkaline treatment. ** Reference 6 is an erratum for reference 5.
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 36917 EPR STUDIES OF P R O T E I N STATE T R A N S I T I O N S IN C H R O M A T I U M F E R R I C Y T O C H R O M E c'
MARTIN M. MALTEMPO* Department of Physics, Columbia University, New York, N.Y. 10027 and IBM Thomas J. Watson Research Center, Yorktown Heights, N.Y. 10598 (U.S.A.)
(Received April 25th, 1974) (Revised manuscript received September 16th, 1974)
SUMMARY Recent magnetic studies have shown that in the pH range 1 to 11 the bacterial heme protein ferricytochrome c' can undergo reversible transitions between various pure high-spin and quantum mechanically mixed (intermediate and high)-spin protein states. The EPR data presented here extend the recent work to high alkaline p H and show that above pH 11.6 reversible transitions occur between various pure high-spin and pure low-spin protein states. The new data and the previous magnetic studies of the protein are discussed in the context of the entatic nature of the iron-porphyrin complex in ferricytochrome c'. EPR data are also provided concerning an anomalous, but reversible, transition to a protein state with reduced heme iron, triggered by freezing and thawing at physiological pH.
INTRODUCTION Recently, it has been shown that in the pH range 1 to 11 the heme protein, ferricytochrome c', isolated from the photosynthetic bacteria Cbromatium, can undergo reversible transitions between four different protein states, two high-spin states and two states corresponding to quantum mechanical admixtures of intermediate and high-spin states [1]. As such, ferricytochrome c' has been proposed as a particularly interesting example of a protein in an "entatic" or "poised" state [1 ]. The EPR studies reported here for Chromatium ferricytochrome c' support this view, indicating that above pH 11.6 the protein undergoes reversible transitions to additional high- and low-spin states. The magnetic properties and pH dependence of these states will be discussed in the context of previous magnetic studies at high alkaline p H [2], as well as the studies performed at physiological and acidic pH [1,2]. We will also discuss EPR results for ferricytochrome c' at pH 8.6 which indicate that a reduced iron complex can be stabilized by freezing and thawing of the protein. * Current address: Johnson Research Foundation, School of Medicine, University of Pennsylvania, Philadelphia, Pa. 19174, U.S.A.
96 METHODS
The isolation and purification of the Chromatium cytochrome c' used in this study has been described in previous publications [1,3]. All measurements at alkaline pH were made on protein in mixed-ion buffer 200 mM in each of glycine, sodium acetate, sodium phosphate and Tris. The protein used in the EPR studies at pH 8.6 was in a 25 mM buffer, with the same ion constituents as used in the alkaline measurements. Protein concentrations were determined from optical absorption measurements made using a Cary model 14 spectrophotometer, assuming a molar heme extinction coefficient of 84.4 mM -1.cm -1 for the 400-nm Soret band of the oxidized protein at pH 7.0. EPR measurements were made with a Varian E-9 X-band spectrometer. Protein concentrations were in the range 0.3 to 1.0 mM in heine iron. The protein samples were cooled to as low as 7 °K by liquid helium gas flow, as described elsewhere [1 ]. RESULTS
The high alkaline pH EPR measurements reported here for Chromatium ferricytochrome c' were made on four different protein samples, obtained from two different protein preparations. In each case the protein samples were first monitored by EPR in the pH range 7 to 11, where each sample showed the pH-dependent interconversion between protein states As, A2 and B2, characteristic of ferricytochrome c' [1]. The pH of the protein was then increased slowly above pH 11.0 by dialyzing the protein overnight against buffer at 4 °C. No change in the EPR spectrum of the protein was observed until the pH of the protein was increased to 11.7, where a sharp spectral transition was observed. Fig. 1 shows the EPR spectrum of one of the protein samples at pH 11.7 and 21 °K, before the transition has occurred, where the protein still has an EPR spectrum characteristic of the high-spin state B2*. The sample shown in Fig. 1 was then thawed,
pH 11,7AND 21°K, PRIOR TO HIGHALKALINE SPECTRAL TRANSITION
# I100
22'00 -
53'00
MAGNETIC FIELD (gouss)
Fig. 1. EPR spectrum of Chromatium ferricytochrome c' at pH ll.7 and 21 °K, prior to the high alkaline spectral transition. The microwave power was 10.0 roW; the frequency was 9.18 GHz. * The EPR spectra shown in this study contain an isotropic signal at about 1525 G, i.e. g .... 4.28. Resonances occurring at this value of magnetic field correspond to paramagnetic species in environments of purely rhombic symmetry and are quite common impurities in iron protein.
97 ~
'°tit
pH 11.7AND 15" K,
A TER T.E .,GH C3
I100
cC3_z B
2200 , MAGNETIC FIELD (gauss)
33~00
Fig. 2. EPR spectrum of Chromatium ferricytochrome c' at pH 11.7 and 15 °K, after the high alkaline spectral transition. The microwave power was 10.0 roW; the frequency was 9.18 GHz.
d i a l y z e d against buffer for 2 m o r e days at 4 °C a n d refrozen. T h e p H o f the s a m p l e was again m e a s u r e d to be a b o u t 11.7, b u t a new E P R s p e c t r u m was o b t a i n e d at 15 °K, as shown in Fig. 2. E a c h o f the other three p r o t e i n samples showed a similar high p H spectral transition. It a p p e a r s that the spectral t r a n s i t i o n is either slowly time d e p e n d e n t , o r it is specific to a quite n a r r o w p H range near 11.7. T h o u g h some o f the p a r a m a g n e t i c signals present in Fig. 2 are p a r t i a l l y s a t u r a t e d at 10 m W m i c r o w a v e power, extensive E P R m o n i t o r i n g o f ferricytoc h r o m e c' in the p H range 11.7 to 12.4 indicates that the t r a n s i t i o n at p H 11.7 results in five new E P R spectral states: B2' , B3, C1, C 2 a n d C3. The three spectral c o m p o n e n t s c o m m o n to each state were identified on the basis o f t e m p e r a t u r e - d e p e n d e n t b r o a d e n i n g , as well as p H a n d s a t u r a t i o n effects. These spectral c o m p o n e n t s are labeled in Fig. 2 a c c o r d i n g to the a p p r o p r i a t e E P R spectral state. The five h i g h - p H spectral states can be listed in o r d e r o f ease o f saturation as C1 > C2 > C3 ~ B2' ~ B3. The g values associated with each o f the spectral states are listed in T a b l e I, along with the g values o f the previously discussed p r o t e i n
TABLEI EPR g VALUES OF FERRICYTOCHROME c" PROTEIN STATES Protein state
g values a
pH b ( ~ amount)
A~ A2 Bt B2 B2"
4.75, 1.99 [1] 5.27, 1.99 [1] 5.94, 2.02 [1] 6.14,5.68,2.0011] 6.16, 5.73, 1.99 7.06, 4.73 ± 0.02 c 2.45, 2.07, 1.98 2.94, 2.23, 1.66 3.23, 2.31 c 2.022, 2.017, 2.004 d
7.0 (98%) [1] 10.0 ( ~ 33~) [1] 1.0 (> 99~) [i] 11.0(>99%)[1] 1t.7+ I 1.7 + 11.7-11.9 12.4 11.7+ 8.6
B3 C1 C2 C3 D
=g values are accurate to !0.01, unless otherwise indicated. h The pH at which there is the largest number of ferricytochrome c" molecules in a particular protein state (pH 11.7+ - pH where high alkaline spectral transition has occurred). c Existence of third EPR signal component uncertain. d The g values pertain to a hyperfine-split free radical signal.
98 states of ferricytochrome c':Ba (pH 1), A1 (pH 7), A2 (pH 10), and B2 (pH l l) [1], For an anisotropic signal monitored in the pH range 11.7-12.4, the g values of the EPR derivative signal were assigned to the low-field maximum, the high-field minimum, and the mid-field zero-crossing. Care was exercised to monitor the various EPR spectral species at values of pH and microwave power when neighbouring species were either absent or power saturated. Though the EPR signals of protein state B2 and spectral state B2' are similar, there are differences in the temperature-dependent line shape of the two EPR signals. In the present studies, as well as previous work [1 ], the rhombic splitting of the EPR signal of protein state B2 in the region of g -- 6 was clearly visible as two distinct peaks below 40 °K. However, the high-field component of the rhombically split signal of State B2' (in the region o f g = 6) is visible only as a shoulder, even in spectra obtained at 16 °K. Though Table I indicates that there is a small difference in the apparent g values associated with the two EPR signals, the significance of this measurement is uncertain due to the different temperature-dependent line shape of the two signals. After the sharp transition depicted in Fig. 2 has taken place, EPR spectra indicate that there are comparable amounts of protein in each of the high-pH EPR spectral states. As the pH of ferricytochrome c' is increased above 11.7, the intensity of signals B2', Ba and C3 decrease, until at pH 12.4 almost all of the protein ( ~ 95 ~o) has been converted to EPR spectral states Ca, and C2. Fig. 3 shows the EPR spectrum of ferricytochrome c' obtained at pH 12.4 and 31 °K, using 1.0 mW microwave power. The EPR data indicate that each of the five high-pH spectral states has a different, reversible pH titration curve. A close examination of Fig. 1 shows that a small, but measurable, amount of protein has been converted to EPR spectral state Ba, in the absence of any observable amount of protein in the other high-pH spectral states. A small B 3 EPR signal was also observed in spectra taken at pH l l.4, but not in spectra obtained at pH ~ 11.2. When the pH of ferricytochrome c' was decreased below l l.7, the protein changed reversibly back to protein state Bz and, if the pH was lowered further, to protein states A1, A2 and Ba. However, it was found that the backward transitions from the high-pH spectral states, C2, C1, C3, Bz' and Ba, down through protein states
C2
,
C3C2
pH 12.4AND 31° K
.60
2~oo
3~oo
MAGNETIC FIELD (gauss}
Fig. 3. EPR spectrum of Chromatium ferricytochrome c" at pH 12.4 and 31 °K, using 1.0 mW microwave power. The microwave frequency was 9.18 GHz.
99 B2, A1, A2 and B1, usually occurred at somewhat lower values of pH than the corresponding forward transitions. Protein samples in this study which showed this hysteresis property (after exposure to pH _>--11.7 for at least 24 h) were found to largely lose this property if the samples were "recycled" twice by titrations between pH 7 and p H 10. It should be briefly noted that in one of the samples used in this study, an anomalous, but reversible EPR spectral transition was observed upon freezing and thawing the sample at pH 8.6, prior to titration at high alkaline pH. The new spectral state, D, was characterized by an orange-red color (as compared with the normally light brown color of the protein at physiological pH), and an EPR spectrum in which there was a narrow 3-fold split paramagnetic signal in the region of g = 2, and a small paramagnetic iron signal in the region of g ---- 6 (reduced to less than 0.5 ~ of the signal observed prior to thawing). The characterization of the signal near g = 2, in spectral species D, was hindered by the presence of an overlapping background signal from a cavity contaminant. However, three sharp minima could be clearly observed at g = 2.002, 2.017 and 2.022, separated by about 16 G. Unfortunately, thawing this sample after the EPR run resulted in a complete spectral and pigment transition of the protein back to the "normal" protein states As, A2 and B2 [1], preventing any further characterization of spectral state D. In the succeeding pH titration from pH 7 to pH 12, this protein sample showed EPR spectra typical of other samples of ferricytochrome c'. There was no further observed change in color, and no appearance of the anomalous EPR signal in the region of g ----2. PROTEIN STATE TRANSITIONS pH~
! •o. PROTEIN STATE KEY A=QUANTUM MECHANICALLY MIXED INTERMEDIATE AND HIGH SPIN B = HIGH SPIN C= LOW SPIN D = REDUCED IRON
/J.~, TRANSITION KEY =pH DEPENDENT ......... FREEZING AND THAWING
Fig. 4. Protein state transitions in Chromatium ferricytochrome c'. The midpoint pH values of some of the transitions are indicated [1] (Maltempo, M. M., unpublished). The number of protein states monitored in the pH range 11.7-12.4, made a determination of midpoint pH values for these transitions difficult.
The observed EPR spectral transitions of Chromatium ferricytochrome c', including those previously reported [1] are shown schematically in Fig. 4. It should be noted that there was no indication in our magnetic studies that the two hemes in the Chromatium ferricytochrome c' dimer are in any way magnetically inequivalent.
100 DISCUSSION The data presented here indicate that above pH 11.7 Chromatium ferricytochrome c' undergoes reversible transitions between five magnetically distinguishable protein states, two high spin and three low spin. In addition, EPR data obtained at pH 8.6 indicate that a reduced iron complex can be stabilized in the oxidized protein by freezing and thawing. These matters will now be discussed in detail. Since each of the high alkaline pH spectral states has a different pH titration curve, we can conclude that each corresponds to a chemically distinct Chromatium ferricytochrome c' protein state. That is, in the temperature and pH range considered, the data presented here rules out the possibility of thermal mixtures of the various magnetic species, arising from ground and excited electronic configurations of the same heme-iron complex. A comparison of the g values and line shape of the various high alkaline pH EPR signals and typical high- and low-spin ferric heme EPR signals [4-6], indicates that protein states B 2' and B 3 are high-spin states and protein states Cj, C 2 and Ca are low-spin states. The symmetry at the iron site in protein states B 2' and B3 is essentially axial, as in other ferric high-spin heme-iron configurations, with the EPR g values indicating 15~o rhombicity [4] in state B 3 and 2 . 7 ~ in state B2'. The EPR g values indicate that the low-spin protein states can be listed in the order of increasing spatial anisotropy at the iron site as: Ca < C2 < Ca. It was pointed out in the preceeding section that while the EPR spectrum of protein state B E has a distinct rhombic splitting in the region of g ~ 6 below 40 °K, rhombic anisotropy in the EPR spectrum of state 12' is manifested only as a highfield shoulder on the g ~ 6 signal, even at 16 °K. The difference between the EPR signals of protein states B 2 and B 2' can be explained by assuming that after the titration of some protein group at pH 11.7, either the relaxation properties of ferricytochrome c' are altered, or more likely, steric strain at the iron site is somewhat "relaxed", resulting in an inhomogeneity in the rhombicity of the protein [7] and a less distinct rhombic splitting in the EPR spectrum. As discussed in the previous section, except for the presence of a small amount of protein in State B3 (Fig. 2), all of the molecules of ferricytochrome c' are in protein state B E until the sharp transition at pH 11.7; whereupon protein molecules are monitored in States B2', C1, C 2 and Ca, as well as in State Ba. However, it would seem quite unlikely that a number of sharp transitions from protein state B2 would all occur at about the same value of pH. Moreover, it was noted that during titratiom from physiological pH to high alkaline pH, the EPR spectral transitions to States C~, C2 and C3 were never observed before the appearance of signal I]2' , and that as the pH was increased above l l.7, the intensity of signals C1 and C2 increased at the expense of signal B2'. These data, taken in conjunction with the similarity in the magnetic properties of States 8 2 and B2', suggest that protein state B 2' may be a metastable intermediate bridging protein state B2 and the low-spin states. In this scheme, protein molecules can undergo a transition from protein state B2 to either the metastable protein state B 2' or protein state B3, but not directly to any of the low-spin states. However, as soon as the transition from Bz to B 2' has taken place, protein molecules can undergo transitions to the various low-spin states. This scheme is consistent with the EPR
101 data and avoids the alternate, unlikely explanation that there are a number of sharp transitions occurring with the same midpoint pH values. Other workers monitoring Chromatium and R. rubrum ferricytochrome c' by EPR, have previously reported the appearance of a new high-spin state at pH 11.6, followed by a transition to a low-spin state as the pH increased above 11.6, though no details were provided concerning the g values or line shape of the signals [2]. The transition to the low-spin state was reported to be complete at pH 12.1 in the R. rubrum protein, but still incomplete in the Chromatium protein at pH 12.0. The earlier study is in general agreement with our data; however, in the absence of detailed EPR data in the previous work it is unclear which two alkaline states were monitored. Our data indicate that at pH 12.4 almost all (=> 95 ~ ) of the protein has been converted to the low-spin states C~ and C2. Previous work, in which Chromatium ferricytochrome c' was monitored by optical absorption spectroscopy, has shown that the spectral transition from protein state B2 to the high alkaline pH spectral states is completely reversible when the pH is neutralized within a few minutes of the forward transition (Cusanovich, M. A., unpublished). In the context of the previous work, the observation of a pH hysteresis effect in our alkaline-treated protein samples may be a result of our method of pH titration, in which protein samples were dialyzed overnight against buffer at 4 °C, or simply the ability of EPR to provide more specific iron-heine magnetic data than optical absorption spectroscopy. Alkaline-treated protein samples which showed the hysteresis property were found to largely lose this property after being "recycled" twice by titrations between pH 7 and pH 10. EPR spectral State D, monitored in one of the protein samples at pH 8.6 prior to alkaline treatment, has not been observed in any of our previous studies [1]. The absence of a strong EP R iron signal suggests the possibility that protein aggregation has occurred, resulting in excessive dipole broadening of the paramagnetic iron signal. However, this possibility must be considered unlikely in view of the low protein concentration, the appearance of the g ~ 2 signal, and the complete reversibility of the transition to State D. It seems more likely that the iron ion has been reduced in State D, and that the protein state is a genuine, if somewhat anomalous, state of cytochrome c'. This view is supported by the EPR spectra of that sample in the pH range 7 to 12, which (except for the observed transition to State D) are typical of other sample spectra of ferricytochrome c'. The g values, line width and saturation properties of the g ~ 2 EPR signal indicate that it is probably a hyperfine-split free radical signal. The 3-fold splitting of the signal, and the apparent reduced state of the iron ion, would alone suggest the possibility that the transition to State D is accompanied by reversible charge transfer from, in all likelihood, an axial nitrogen ligand (I = 1), to the iron ion. However, this interpretation must be considered quite speculative, since a rough calculation indicates that the g ~ 2 signal in State D represents at least an order of magnitude less electron spin than that represented by the iron signal prior to the protein state transition. In summary, the data presented here, in conjunction with previous work [1], show that Chromatium ferricytochrome c' can undergo reversible transitions between ten magnetically distinguishable protein states. Nine protein states have oxidized heme iron: four (Ba, B2, B2' and Ba) are high spin, three (Ca, C2 and Ca) are low spin, and two (A~ and A2) are quantum mechanical admixtures of intermediate and
102 high-spin states [1]. The reversible transitions between these nine protein states are pH dependent. The remaining protein state, D, is somewhat anomalous, having been monitored in only one sample of ferricytochrome c'. EPR data for State D indicate that the iron ion is probably reduced, perhaps stabilized by reversible chargetransfer to the iron ion from an axial nitrogen ligand, triggered by freezing and thawing. It has been suggested that the quantum mechanically mixed-spin ferric heine complexes which predominated in ferricytochrome c' at physiological pH can be regarded magnetically (and perhaps structurally, as well) as "bridges" between the high- and low-spin ferric heme complexes [8]. The ability of ferricytochrome c' to undergo reversible transitions between these unique mixed-spin states and various high- and low-spin states provides new evidence for the entatic nature of the iron-porphyrin complex. It is hoped that future work will consider the role which the various protein states of ferricytochrome c' play in the biological function of the protein*, and whether there exists a correlation between the magnetic properties of the various states and the corresponding chemical properties, such as the redox potential. There is also the very interesting question of what role the ferricytochromes c' played in heme protein evolution. The iron-heme structural configuration inferred from the magnetic data at physiological pH [1] suggests the possibility that these unusual cytochromes may be evolutionary intermediates between high- and low-spin heme proteins. ACKNOWLEDGEMENTS The author wishes to thank Ms Edith R. Shapiro for her valuable technical assistance in the preparation and purification of Chromatium cytochrome c', and Dr Thomas H. Moss for innumerable, helpful discussions. REFERENCES 1 Maltempo, M. M., Moss, T. H. and Cusanovich, M. A. (1974) Biochim. Biophys. Acta 342, 290-305 2 Ehrenberg, A. and Kamen, M. D. (1965) Biochim. Biophys. Acta 102, 333-340 3 Cusanovich, M. A. (1967) Ph. D. Thesis, University of California at San Diego, U.S.A. 4 Peisach, J., Blumberg, W. E., Ogawa, S., Rachmilewitz, E. A. and Oltzik, R. (1971) J. Biol. Chem. 246, 3342-3355 5 Salmeen, I. and Palmer, G. (1968) J. Chem. Phys. 48, 2049-2052 6 Salmeen, I. and Palmer, G. (1968) J. Chem. Phys. 48, 4331"* 7 Mailer, C. and Taylor, C. P. S. (1972) Can. J. Biochem. 50, 1048-1055 8 Maltempo, M. M. (1974) J. Chem. Phys., 61, 2540-2547
* Though cytochrome c' is generallythought to be involvedin the cytochrome electron-transport system in bacteria, the specific function of the protein is unknown. The possibility that a protein state monitored in the isolated protein at acidic or alkaline pH might be biologically active in vivo should not be excluded, since it is conceivable that these states could be stabilized by conditions other than acidic or alkaline treatment. ** Reference 6 is an erratum for reference 5.