Magnetic studies on the changes in the iron environment in Chromatium ferricytochrome c′

Biochimica et Biophysica Acta, 342 (1974) 290-305

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 36678 M A G N E T I C STUDIES ON T H E C H A N G E S IN T H E IRON E N V I R O N M E N T 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 a, THOMAS H. MOSSa and MICHAEL A. CUSANOVICH b aDepartment of Physics, Columbia University, New York, N.Y. 10027 and IBM Thomas J. Watson Research Center, Yorktown Heights, N.Y. 10598, and bDepartment of Chemistry, University of Arizona, Tucson, Ariz. 85721 (U.S.A.)

(Received August 24th, 1973) (Revised manuscript received December 27th, 1973)

SUMMARY Cytochrome c', a heme protein isolated from photosynthetic and denitrifying bacteria, has previously been shown to have unusual chemical and physical properties. EPR and magnetic susceptibility measurements reported here indicate that in the pH range 1-11, the oxidized form of the protein can exist in four magnetically distinguishable states. Reversible transitions between these states can be induced by changing the pH of the protein solution. The two protein states which exist at physiological pH have magnetic properties unlike any other known heme protein. We show that these unique magnetic properties can best be explained by postulating iron electronic states which are quantum mechanical admixtures of an intermediate spin state and a high spin state. The suggestion, previously made, that the unusual magnetic properties of the protein are due to a thermal mixture of high and low spin states, is shown to be inconsistent with the magnetic data. The protein states at pH 1 and 11, though slightly dissimilar in symmetry at the iron site, are both typical ferric high spin states, quite similar in magnetic properties to the acid forms of metmyoglobin and methemoglobin. In the pH range 1-11 small anions are unable to bind to the iron site, indicating the presence of a strong hydrophobic region in the vicinity of the heme. Possible ligand-iron configurations corresponding to the four different protein states are discussed.

INTRODUCTION Metallo-proteins, and in particular heme proteins, perform a wide variety of biological functions and have diverse chemical and physical characteristics. Physical probes such as EPR, Mossbauer spectroscopy, and magnetic susceptibility measurements have often been used as aids to determine the electronic state at the heme site with the goal of relating the structure of the protein at its active site to its chemical function. We wish to report physical studies of the bacterial protein cytochrome c', which offer the possibility of new insight into the mechanism by which subtle differences in the structure of proteins are manifested in their different chemical properties.

291 The cytochromes c' are particularly interesting in this regard, in that they have ligand binding and structural characteristics similar to the c-type cytochromes, but magnetic and spectral properties closer to those of hemoglobin and myoglobin. Cytochrome c' has been extracted from the denitrifying bacteria Pseudomonas denitrificans [1] and from a variety of photosynthetic bacteria (refs. 2-7 and Meyer, T., Cusanovich, M. A. and Kamen, M. D., unpublished). Most of the cytochromes c' isolated to date are diheme proteins of 24 000-30 000 molecular weight [8]. However, a monoheme cytochrome c' of 14 000 molecular weight has been isolated from Rhodopseudomonas palustris [9, 10]. Most of the diheme cytochromes c' can be dissociated into monoheme subunits in the presence of the proper agents [8]. Recent work indicates that the two monoheme peptides obtained upon the dissociation of the Chromatium protein have the same amino acid compositions [11]. Consequently, it appears likely that the isolated diheme cytochromes c' are true dimers*. At room temperature and physiological pH the visible absorption spectrum of cytochrome c' is similar to those of hemoglobin and myoglobin, having absorption peaks at 490 and 635 nm in the oxidized form, and a broad peak centered at about 550 nm in the reduced form. However, EPR, Mossbauer and magnetic susceptibility data taken at physiological pH have shown that there are significant differences between the nominally high spin state of the iron ions in ferricytochrome c' and the electronic state of the iron ion in typical ferric high spin heme proteins such as acid methemoglobin and acid metmyoglobin [15-17]. In addition, ligand-binding experiments with Rhodospirillum rubrum ferricytochrome c' in the pH range 5-12 show that the protein does not react with simple anionic ligands such as F-, N3-, or CN- [18], although these are known to bind readily to ferrihemoglobin and ferrimyoglobin, In this respect cytochrome c' resembles the native form of cytochrome c more than hemoglobin or myoglobin. Also, as with cytochrome c, the heme moiety of cytochrome c' is bonded, at positions 2 and 4 of the porphyrin ring, to cysteinyl residues on the protein peptide chain [11]. Unlike cytochrome c, however, reduced cytochrome c' will readily bind CO at the iron site [18]. Both the oxidized and reduced forms of cytochrome c' have previously been shown to exist in different molecular states. These states have been characterized by optical absorption [14, 19] and CD spectroscopy [19], as well as the kinetics of CO binding [20, 31 ]. Transitions between the different states can be induced by changing the pH of the solvent or by the addition of organic solvents. The studies reported in this paper on Chromatium ferricytochrome c' analyze the pH-induced changes of the protein by monitoring specifically the iron site, in order to see whether the functional and spectroscopic changes previously observed are related to the changes in the electronic configuration of the heme moiety. METHODS The Chromatium cytochrome c' used in this study was isolated and purified according to procedures previously reported [22]. Four separate protein preparations were used, yielding protein with a purity index [11] in the range 0.34-0.36. Protein * A cytochromec'-like heme protein of molecular weight 170 000, recently isolated from the bacterium Azotobacter vinelandii [13], may represent a polymer of cytochromesc'.

292 purity was also monitored by acrylamide gel electrophoresis [23, 24]. Fig. 1 shows the gel electrophoresis obtained before the protein passed through the final D E A E column (gel tube 2), after the protein passed through the final D E A E column (gel tube 1) and after the final protein sample was concentrated (gel tube 3). The figure shows that there is a substantial increase in the purity of the protein due to the final D E A E chromatography. The reader will also note that the relative intensity of the minor band seen in gel tube 1 decreased substantially upon concentration of the protein (gel tube 3). No further chromatography was performed between the gel electrophoresis of Tubes 1 and 3; the protein was merely concentrated by ultrafiltration using a membrane retaining molecules of molecular weight greater than 10 000. In another protein preparation, following the final D E A E chromatography, it was found that a minority protein species with a cytochrome c'-type optical spectrum was passing through a 10 000-molecular weight filtration membrane, but could be contained by a 1000-molecular weight filtration membrane. This indicates that the minor band in gel tube l of Fig. l probably

Fig. 1. Acrylamide gel electrophoresis performed during isolation and purification of ferricytochrome c' from Chromatium cells. The figure shows the gel electrophoresis obtained before the protein passed through the final DEAE column (gel tube 2), after the protein passed through the final DEAE column (gel tube 1) and after the final protein sample was concentrated (gel tube 3). The lower gel buffer was 12~ in acrylamide at pH 8; the upper gel buffer was 2.5~ in acrylamide at pH 9. The amount of protein used varied from about 60 to 1500 pmoles. represents a monoheme peptide Of cytochrome c' with a molecular weight close to 10 000. All measurements reported here were made on protein in mixed-ion buffer 25 m M in each of glycine, sodium acetate, sodium phosphate and Tris. Protein concentrations were determined from optical absorption measurements made using a

293 Cary model 14 spectrophotometer, assuming a molar heme extinction coefficient of 84.4 raM-1, c m - t for the 400-nm Soret band of the oxidized protein at p H 7.0. EPR measurements were made with a Varian E-9 X-band spectrometer. Protein concentrations were in the range 0.3 -1.0 m M in heme iron. The protein samples were cooled to as low as 7 °K by liquid helium gas flow. Protein temperatures were monitored by recording the resistance of a calibrated carbon resistor mounted near the sample. The magnetic moment of the protein was obtained from magnetic susceptibility measurements made between 4.2 and 1.4 °K, using magnetic fields of 2 kG and below. In the field range considered the bulk magnetization was linear in magnetic field. Protein concentrations were between 0.9 and 1.3 mM in heme iron. The magnetic susceptometer used in this study has been described elsewhere [26]. In order to extract magnetic parameters from the EPR spectra, a computer program was written to simulate the spectra at various temperatures. The program was written for an axially symmetric paramagnetic species arranged in a powder distribution, assuming the existence of a Lorentzian line shape. Consideration was made for the variation in transition probability as a function of the geometrical orientation of the spins with respect to the microwave field. An exact closed-form solution was obtained for the derivative of the absorption spectrum*. The function which we used for the transition probability is strictly correct only for a spin Hamiltonian in which the electronic Zeeman term is the dominant term. Though the Zeeman term is not the dominant term in the spin Hamiltonian for cytochrome c' (the crystal field term is), the small error implied in our choice of the probability function does not significantly affect our results**. RESULTS EPR spectroscopy at low temperatures, in the p H range 1-11, indicates that the oxidized form of cytochrome c' can exist in three distinguishable EPR spectral states. Fig. 2 presents EPR spectra, labeled B1, A and B2, which correspond to these three different states, observed at 77 °K and below 20 °K. Transitions between these different spectral states can be induced by changing the pH of the protein solution. All of the transitions are reversible. The EPR data indicate that at physiological pH almost all ( > 9 9 ~ ) of the protein molecules are in EPR spectral state A***. The line shape of the EPR spectrum at 77 °K is quite broad; it is only at temperatures below about 30 °K that many details of the spectrum become clear. The apparent g values of the spectrum are 4.77 and 1.99. These g values were taken from the derivative of the absorption spectrum obtained at 7 °K: the value o f g x was obtained from the zero-crossing in the region of * An equivalent solution, developed independently, has previously been reported [26]. For a single paramagnetic species with g ± = 6 and g ii = 2, one probability function changes by a factor of 0.56 in going from gil to g±, while the other function changes by a factor of 0.50 in that region. This corresponds to a 10~ difference in the average field derivative of the two probability functions. This matter will be discussed in detail in a later paper. *** At physiologicalpH the low field shoulder of the EPR spectrum atg ~ 6 is probably due to the presence of a small amount of protein in States B1 and B2. The relative population of each EPR spectral species was obtained from the EPR computer simulation program. **

294

~

BI, pH 0.9 AND II°K BI, pH 1.0 AND 73°K

y

f

>)-

z_.

A, pH 7.2 AND 77°K

awa B2, pH 10.8 AND 17°K B2, pH 10.8 AND 82°K

1~--~1 I I00

2500 3300 MAGNETIC FIELD (gauss)

,,oo

Y

t

r

2500 :5300 MAGNETIC FIELD (gauss)

Fig. 2. (a) EPR spectra of Chromatium ferricytochrome e' at liquid nitrogen temperatures, in the pH range 1-11. The microwave frequency was 9.17 GHz. (b) EPR spectra of Chromatium ferricytochrome c' below 20 °K, in the pH range 1-11. The microwave frequency was 9.17 GHz. g = 4.8, while the value ofgll was o b t a i n e d f r o m the m i n i m u m in the region o f g = 2*. T a b l e 1 lists the g values o f E P R s p e c t r u m A a l o n g with the values o f the line width at 7 a n d 77 °K, as m e a s u r e d by the difference in field between the two extrema at g~5. M a g n e t i c susceptibility m e a s u r e m e n t s show t h a t in the t e m p e r a t u r e range from 4.2 to 1.4 °K the effective p a r a m a g n e t i c m o m e n t o f the p r o t e i n in E P R spectral state A has a value o f a b o u t 3.4 #B ( B o h r magnetons). T h o u g h f e r r i c y t o c h r o m e c' has been generally considered to be in a high spin state at p H 7, the values o f the m a g n e t i c susceptibility a n d the E P R p a r a m e t e r s listed in T a b l e I for E P R spectral state A are c o n s i d e r a b l y different f r o m the c o r r e s p o n d i n g values for a typical high spin iron p r o t e i n like acid m e t m y o g l o b i n . In addition, the E P R line shape changes r a p i d l y with t e m p e r a t u r e in the 77-40 °K range, a behavior n o t n o r m a l l y observed in high spin heme proteins. As the p H o f the p r o t e i n is lowered to 3.0, m o r e t h a n 9 9 ~ o f the protein molecules are still in E P R spectral state A. However, as the p H o f the p r o t e i n is decreased further, f e r r i c y t o c h r o m e c' undergoes a transition to a different E P R spec* The assignment of g values from an EPR spectrum is an ambiguous matter. Using our EPR simulation program we were able to show that the g values of an axial signal will, in general, not correspond to either the maximum or minimum of the spectrum or to the zero-crossing. The g values can only be obtained from an accurate simulation of the spectrum, which we describe in the next section. The apparent g values described here are useful in characterizing the EPR spectrum, and correspond to the location of the peaks and shoulders of the absorption spectrum (the integral of the EPR spectrum).

295 tral state; the EPR spectrum corresponding to this new state is labeled B1 in Fig. 2. The midpoint of this transition was found to be pH 1.5 (4- 0.1), based upon computer simulations of EPR spectra at pH 2.4, 1.gand 1.3. At pH 1.3 the spectral transition is complete; all of the protein molecules are in EPR spectral state B1. The apparent g values of EPR spectrum B1, obtained from the derivative of the absorption spectrum at 11 °K are 5.94 and 2.02. At 73 °K the line width parameter, AH, has a value of 70 G, while at 10 °K the value of the width parameter has decreased to 45 G. Magnetic susceptibility measurements show that in the temperature range from 4.2 to 1.4 °K the effective paramagnetic moment has a value of 4.7 #B. AS the pH of the solution of ferricytochrome c' is increased one observes the reversible transition from EPR spectral state B~ back to EPR spectral state A. As the pH is increased above pH 7.4 a second spectral transition is observed, going from EPR spectral state A to the spectral state labeled B 2 in Fig. 2. The midpoint of this transition was found to be pH 9.8 (4- 0.2), based upon computer simulations of EPR spectra at pH 9.4, 9.5, 9,9 and 10.0. At pH 10.4 the spectral transition is complete; all of the protein molecules are in EPR spectral state B2. The apparent g values of EPR spectrum 8 2 a r e 2.00, 5.68 and 6.14. The criteria used to assign g values in the region of g = 6 was to take one g value at the extrema of the absorption derivative which was lowest in magnetic field and the second g value at the zero-crossing. The value of g iF was obtained, as before, from the minimum in the region of g = 2. At 76 °K the line width, AH, has a value of about 80 G, while at 17 °K the line width has decreased to 50 G. The values of the line width were obtained by taking the difference in field between the high field maximum in the region of g = 6 and its corresponding minimum. Magnetic susceptibility measurements show that at pH 10.0, in the temperature range from 4.2 to 1.4 °K, the effective paramagnetic moment of the protein is 4.3 #B. Above pH 11.6 EPR and optical transitions were observed from State B2 to various high and low spin states. Our results on these states, two of which have previously been discussed [15, 19], will appear in a later paper. The EPR spectrum of State A, recorded at 77 °K (Fig. 2a), shows a more complicated signal in the region of g = 2 than the spectrum observed at 7 °K (Fig. 2b). The high temperature spectra consist of a superposition of two signals in the region of g = 2: the signal due to the heme iron and a signal due to a minor copper contaminant*. The signal of the copper contaminant is not seen in the spectra taken at low temperatures; the copper signal is completely saturated leaving a clear, unaffected iron signal. Some of the spectra in Fig. 2 also contain a small 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. EPR spectroscopy indicates that the transitions between the spectral states B1, A and B 2 of ferricytochrome c', which occur during pH titration, are completely reversible. Spectral states A and B 2 w e r e studied in four separate protein preparations, and State B1 in two preparations. All of the above EPR data were reproducible in the different protein preparations. R o o m temperature optical absorption spectra were obtained in the pH range 1-10.5. All spectra showed an asymmetric Soret band peaked between 396 and 408 * The copper contaminate could not be removed by dialysis against EDTA.

296 nm with a shoulder at about 375 nm, and secondary maxima at 500, 535 and 635 nm. The relative intensity of these peaks changed with pH titration ; the relative intensity at 635 nm was a minimum at physiological pH. The interaction of various anions with ferricytochrome c' was studied to determine whether the differences in the electronic state of the iron ion in spectral states B~, A and 82, corresponded to differences in anion accessibility to the iron site. The room temperature optical absorption spectrum of ferricytochrome c' was monitored in 100 mM mixed-ion buffer at pH l, 7 and l0 in the presence of NaF, NaC1, NaN3 and KCN. The optical spectrum of the protein in the presence of the various anions was unchanged from the native spectrum, up to a 200 molar excess of anion/protein. DISCUSSION At low temperatures, in the pH range 1-11, we have shown that the oxidized form of cytochrome c' can exist in three distinguishable EPR spectral states, and that the transitions between these different states are completely reversible. At pH 1 (spectral state B1) or pH 11 (spectral state Bz) the protein has magnetic properties which are quite similar to those of typical high spin ferric heme proteins, such as methemoglobin and metmyoglobin. However, the protein at physiological pH (spectral state A) has magnetic properties unlike those of any other known heme protein. We will discuss each state separately, including possible iron electronic configurations which might correspond to the highly unusual state at physiological pH. Some arguments concerning the nature of the axial ligands of the iron ion in EPR spectral states B1, A and B 2 will also be inferred from their magnetic properties. Though spectral state A is inherently the most interesting of the three spectral states, due to its unique magnetic properties, EPR spectrum A is also the most complex. In order to obtain useful magnetic parameters from a spectrum with the unusual line shape and temperature dependence of State A it was necessary to simulate the EPR spectra at various temperatures. These simulations show that it is not possible to simulate EPR spectrum A with a single species spectrum. This is true even allowing for a totally anisotropic g tensor, because the components of such a spectrum would have very nearly the same temperature dependence. Simulations of the spectra at 7, 14, 29, 45 and 77 °K indicate that the unusual temperature dependence and line shape of Spectrum A are due to a superposition of two different EPR signals, A1 and A2, which have a temperature-independent weight ratio of 40:1. Fig. 3 shows the EPR spectrum of State A at 7 °K and the corresponding simulated spectrum. Signal A~ has g values of 4.75 and 1.99, and a line width of 350 G, while Signal A 2 has g values of 5.27 and 1.99, and a line width of 75 G. The computer simulations indicate that the symmetry of the g tensor of Signal A2 is essentially axial; if there is a rhombic component of symmetry it is small ( < 5 %). Due to the large line width and broadening of Signal A1 the extent of its rhombicity is uncertain. Our belief that Signal A1 is an anisotropic signal with a broad gtl component near 2 is supported by our magnetic Susceptibility measurements, which are discussed below. The magnetic parameters obtained from the simulation of Spectrum A at 7 °K are listed in Table I, along with the corresponding parameters obtained from the simulation of the spectrum at 77 °K. For computational reasons, the simulated program uses a Lorentzian line shape, and though the simulated spectrum is quite similar to the actual spectrum, the

297 actual spectrum intensity drops off much more rapidly in the wings than does the simulation. Since the wings of a Gaussian line shape decrease more rapidly than a Lorentzian line shape, we expect that a simulation program using a Gaussian line shape would provide better agreement with the actual spectrum at the low field side of the g ~ 5 resonance and at g ~ 2. The g values, line widths and weight factors of

/•

...... ~ . l J ~

I

I100

SPECTRUM - SIMULATION

I

2500 MAGNETICFIELD(gauss)

3300

Fig. 3. EPR spectrum and computer simulation of Chromatium ferricytochrome c' at pH 7.2 and 7 °K. The weight factors of Signals A1 and A2 in the computer simulation are in a ratio of 40:1. Signals A1 and A2 are, to some degree, dependent upon the choice of the line shape used in the simulation program. In Table I, therefore, along with the EPR simulation parameters used in Fig. 3, we have estimated the corresponding uncertainty in the parameters, including the uncertainty due to the choice of line shape. However, independent of the form chosen for the absorption curve, extensive spectral simulations indicate that the unusual line shape and temperature dependence of EPR spectrum A are due to a superposition of two signals with the general characteristics outlined above. The basic chemical and structural question arising from these results is whether the two components of EPR spectrum A arise from: (1) differences between the two hemes in the same protein molecule, (2) the ground and excited states of the heme in the same protein conformation, or (3) protein molecules in two different conformational states with slightly different heme environments. The first possibility can be eliminated since it would require equal weight factors for EPR signals AI and A2, in contrast to observations. The second possibility can also be eliminated on the basis of the temperature independence of the relative weight factors of Signals AI and Az in the temperature range 7-45 °K. Thus, we conclude that EPR signals A1 and A2 arise from protein molecules in two different conformational states with slightly different heme environments. Magnetic susceptibility measurements made at physiological pH and in the temperature range 1.4-4.2 °K are consistent with the magnetic parameters of Signals A1 and Az used to simulate EPR spectrum A at 7 °K. At 7 °K the weight factors of Signals A1 and A2 are in the ratio of 40:1 ; consequently, the effective paramagnetic moment of the protein at low temperatures must be almost entirely due to the paramagnetic moment of the state giving rise to Signal A~. In general, for a multiplet state the spin-only high temperature limit of/~eff is equal t o ~Seff(aeff -~- 1)(g2eff)av "/A#, where Serf and geft are the effective spin and the effective g value of the paramagnetic '

298 species. W i t h ferricytochrome c' at 3 °K, if one takes S~ff equal to ~/2", g± equal to 4.75, a n d g~l equal to 1.99, one obtains a n effective p a r a m a g n e t i c m o m e n t of 3 . 3 / ~ ; this value is quite consistent with the experimental value of 3 . 4 / ~ given in Table I. A s s u m i n g that Signal A1 consists of a nearly isotropic signal at g - - 4.75, a paramagnetic m o m e n t of 4.2 #a would be expected. Thus, the magnetic data are consistent instead with the idea that Signal AI is anisotropic with a b r o a d gz c o m p o n e n t at g = 2. TABLE I MAGNETIC PARAMETERS OF ACID METMYOGLOBIN AND FERRICYTOCHROME c' g values gll

EPR spectral state A Protein state B1 Protein state A1 Protein state A2 Protein state B2 Acid metmyoglobin

1.99 2.02 1.99 1.99 2.00 2.01

Rhombicity

g ,~

4.77 5.94 4.75 5.27 5.68, 6.14 5.92 [27]

unknown <0.5~ unknown small, if present 3~ 0.1 ~ [28]

EPR line 'width AH** L.T.* (G)

77 °K (G)

510 (±5) 45 (±2) 350 (±20) 75 (±5) 50 (±2) 36 [27]

620 (±5) 70 (±2) 375 (±20) 150 (:k 10) 80 (±2) 75 [27]

neff (at 4.2 °K)

3.4 (±0.1) 4.7 (~0.1) 3.4 (±0.I) unknown 4.7 (+0.2) 4.7 [17]

* L.T. = Low temperature measurements were made at 7 °K for States A, AI and Az, at 11 °K for State B1, at 17 °K for State B2 and 1.6 °K for acid metmyoglobin. ** The line widths of Signals A, B~ and B2 were obtained directly from the EPR spectra; while the line widths of Signals A1 and A2 were taken from the line width parameters used in the EPR simulation program. The value of AH listed for EPR spectral state A is a useful characterization parameter but not a true line width, since Spectrum A is a composite spectrum. As previously p o i n t e d out, a c o m p a r i s o n of the magnetic p a r a m e t e r s of protein states A1 a n d A2 a n d those of acid m e t m y o g l o b i n , as listed in Table l, indicates there are significant differences between their respective h e m e - i r o n electronic states. The typical ferric high spin E P R spectrum with g values of 6 a n d 2 arises from an iron electronic state c o r r e s p o n d i n g to the Sz = -+- 1/2 K r a m e r s d o u b l e t of the g r o u n d state (S - - 5/2) sextet. The most likely e x p l a n a t i o n for the u n u s u a l g values of Signals AI a n d A2, is that in b o t h iron electronic configurations there is a q u a n t u m mechanical admixture to the Sz = -+- 1/2 K r a m e r s d o u b l e t of the g r o u n d state sextet; such a n admixture could arise f r o m the excited K r a m e r s doublets within the sextet or from multiplets of a low lying (S = 3/z) quartet**. Substantial a d m i x i n g between the S z z doublets within the (S ~ 5/2) sextet will occur u n d e r two c o n d i t i o n s : (i) if the zero-field splittings between the K r a m e r s doublets is c o m p a r a b l e in m a g n i t u d e to the Z e e m a n interaction, (ii) if the zero-field * To a good approximation only the lowest Kramers doublet is populated at this temperature. Further, since the susceptibility measurements were obtained for magnetic fields below 2000 G, at 3 °K we have k T >>/~aH. Thus, at liquid helium temperatures the effective paramagnetic moment of the protein can be taken as the high temperature limit of/~e~f for the ground state Kramers doublet. ** It is quite unlikely that there would be an admixture to the Sz = ± 1/2 doublet of the ground state (S = 5/2) sextet from an excited (S = 1/2) doublet; the usual mechanism for such an admixture, the spin--orbit interaction, has no first-order matrix elements between these states.

299 splittings between the Kramers doublets is much larger than the Zeeman energy, and if the crystal field at the iron site has a rhombic component comparable in magnitude or greater than its axial component. The first possibility must be considered quite unlikely. In X-band EPR the Zeeman interaction energy is 0.31 cm -1, while the zerofield splittings in ferric high spin heme proteins have always been found to be greater than 10 cm -1. Further, the low temperature magnetic susceptibility measurements indicate that, at least in the electronic state giving rise to the predominant Signal A1, the zero-field splitting between the Kramers doublets must be substantially larger than the Zeeman energy. This is clear from both the temperature dependence of the susceptibility below 4.2 °K (linear in T-l), and the paramagnetic moment value of 3.4 #~, lower than that expected if all doublets of the S = 5/2 sextet were occupied. The second possibility (that a large rhombic component of the crystal field causes admixing between the Kramers doublets of the S -- 5/2 sextet) must, in general, be considered somewhat more likely than the first. Due to the symmetry properties of the porphyrin ring, the iron sites in heme proteins are, to the first order, axially symmetric. The maximum amount of rhombicity observed at the iron site in a ferric high spin heine protein is about 16 %, found in a minority constituent of alkaline-treated ferrimyoglobin at pH 10.1 [29]. It is conceivable that a greater steric or electronic strain at the iron site could result in a crystal field with a rhombic component which was comparable to or larger than its axial component. However, even if such crystal fields were possible, none of the resulting iron electronic states would be consistent with the g values and magnetic susceptibility data associated with Signals A1 and A2 (Maltempo, M.M., unpublished). The unusually low paramagnetic moment and the unusual g values of the EPR. spectrum of ferricytochrome c' at physiological pH strongly suggest the alternate possibility that the heme-iron electronic states giving rise to Signals At and A2 are both quantum mechanical admixtures of the Sz = ± 1/2 Kramers doublets of the (S = 5/2) spin sextet and a low lying (S = 3/2) spin quartet. It is not uncommon to find a thermal mixture of high and low spin states in ferric heine proteins; however, there has been no previous report of any substantial quantum mechanical admixture of spin states. The energy operator for the spin-orbit interaction has matrix elements between the Sz = ± 1/2 Kramers doublets of the spin sextet and a spin quartet; however, the usual energy separation between these states (about 2000 cm -~) generally precludes any substantial quantum mechanical admixing of them. However, as we will discuss later, there are conditions for which we would expect the two (unperturbed) spin states to be separated only by a few hundred reciprocal centimeters, with the possibility of the spin quartet lying below the spin sextet. We would then expect the ground electronic state of the iron ion to consist of substantial admixtures from the two spin states. This model is consistent with the g values associated with Signals A~ and A2, as well as the low temperature paramagnetic moment of the protein at physological pH, assuming (as shown previously) that Signals A1 and A2 arise from two different protein states (Maltempo, M. M., unpublished). The iron electronic state corresponding to Signal A1 would consist of an admixture of 65 % of the intermediate spin state (S = 3/2 ) and 35 % of the high spin state (S = 5/2), while the iron electronic state corresponding to Signal A2 would consist of an admixture of 65 % of the high spin state and 35 % of the intermediate spin state. In both protein states the unperturbed spin states would be separated by about 150 cm-1. The protein state giving rise to Signal

300 A1 would have the spin quartet lying below the spin sextet; while the protein state giving rise to Signal A2 would have the spin sextet lying below the spin quartet. A detailed discussion of the quantum mechanically admixed spin states will be presented in a paper being prepared for publication. Thus, to summarize the discussion of the origin of Signals A1 and A2: (i) we have considered three mechanisms which would induce a quantum mechanical admixture to the Sz :k 1/2 Kramers doublet of the (S = 5/2) spin sextet; (ii) these considerations support the view (suggested by the simulation of the EPR spectra) that Signals A1 and A 2 arise from different heme environments and most probably different protein conformations, in which the iron electronic configurations consist of quantum mechanical admixtures of the spin sextet and a spin quartet. In contrast to the unusual aspects of the p H 7 states, Table I shows that protein states B~ and B2* a r e both typical ferric high spin states, quite similar in magnetic properties to the acid forms of metmyoglobin and methemoglobin. The g values in Table I indicate that the iron site in protein state B1 has pure axial symmetry, whereas the iron site in protein state B 2 has a small rhombic component of symmetry (about 3 ~o)- The effective paramagnetic moment of States B1 and B 2 at liquid helium temperatures, 4.7 Bohr magnetons**, is consistent with an axial crystal field parameter D equal to 15 cm -~. The close similarity of the magnetic properties of States B1 and B 2 with those of acid metmyoglobin, and the fact that a histidine residue is found adjacent to a cysteine residue on the c' heme peptide, suggest the usual role for histidine as the fifth iron ligand in cytochrome c' Crystal field considerations alone would indicate that the sixth iron ligand positions in either of the high spin B states could be vacant as in deoxyhemoglobin, or occupied by a weak field ligand such as water as in acid metmyoglobin. However, the possibility that the sixth iron ligand position in State B 2 is occupied by a water molecule can be eliminated, since protein state B 2 is stable from its first appearance at about p H 7.8 to p H 11.6. If the weak field ligand were water, it would be expected to dissociate somewhere in that region causing another spectral change. The possibility that a O H - , a stronger field ligand, might occupy the sixth iron position in State B 2 must be considered unlikely, since EPR in the range 17-77 °K and magnetic susceptibility measurements at 150 °K [17] show no evidence of the thermal mixture of spin states expected with this strong field ligand. The proposed admixture of spin states at p H 7 implies that the energy difference between the one electron d~2 orbital and either of the three lower orbitals (the degenerate dx~ and dyz orbitals, and the ground state dxy orbital)must be comparable to that found in typical high spin configurations; while the energy difference between dx2y2 and dxy orbitals must be somewhere between that found in typical high and low spin configurations. These considerations suggest that at p H 7 the iron ion * We make no distinction between the "protein states BI and B2", and the "spectral states B1 and B2", since Spectra B1 and B2 are each apparently the result of one paramagnetic chemical species. ** The paramagnetic moment of ferricytochrome c' was measured to be 4.3/~a at pH 10.0, in the temperature range from 1.4 to 4.2 °K. However, computer simulation of the EPR spectrum at pH 10.0, indicates that only two-thirds of the protein molecules are in protein state B2; the remaining third of the molecules are, for the most part, in protein state A2. If we take the paramagnetic moment of protein state A2 to be about 3.5 Pa (g values indicate that the paramagnetic moment of State A2 will be somewhat higher than the moment of State A~), then protein state B2 should have a paramagnetic moment of 4.7 (:k0.2)/~B. The latter is the value listed in Table I.

301 is somewhere between the out-of-plane position presumed for the typical high spin B states and the in-plane position associated with low spin states, with the nitrogen of the histidine imidazole bonded at the fifth ligand position and the sixth ligand position either vacant or occupied by a water molecule. Possibly, during the transition from either of the B states to one of the A states, a steric strain is imposed upon the iron ion via the axial N - F e bond which favors a more nearly planar hemeiron configuration. It may be that this steric strain is enhanced due to the cysteinyl linkages binding the heme rigidly to the peptide chain. The presence of a weak field ligand, such as a water molecule, at the sixth ligand position would help stabilize a more nearly planar heine-iron configuration. These structure arguments will be discussed more fully in a later paper. The interaction of various anions with ferricytochrome c' was studied to determine whether the similarity between the iron electronic states in acid metmyoglobin and States B, and B2 corresponded to a similarity in anion accessibility to the iron site. The results of the ligand-binding experiments indicate that in the pH range of 1-11 neither F - , N3-, C N - or C1- is capable of binding directly to the heme in ferricytochrome c'. It would seem, then, that even in States B1 and B2, in which the iron electronic states are quite similar to that in acid metmyoglobin, the protein conformation at the iron site in ferricytochrome c' is such as to deny anions access to the iron site. A spatial constraint at the iron site could prevent the bonding of anions; or more likely, the charge distribution in the vicinity of the iron prevents the passage of anions to the iron site. Other workers have studied some of the different states of the ferricytochromes c' using various physical techniques and our results can be correlated with the previous work. The previous studies, mostly done at room temperature, have been concerned with two states which probably correspond to our EPR spectral states A and B2, and with other low spin states obtained above pH 11. The fact that a distinct protein state like our State B1 has not been noted earlier is probably due to the similarity in the room temperature optical spectra at pH 1 and 7. The room temperature transition between the pH 7 state (our EPR spectral state A) and pH 11 state (our EPR spectral state B2) has been monitored by optical absorption and CD spectroscopy in several of the cytochromes c' [14, 19, 34]. Previous ligand binding [18] and organic solvent perturbation experiments [19, 34] have suggested that at pH 7 (EPR spectral state A) and pH 11 (EPR spectral state B2) there is a hydrophobic region in the vicinity of the heine which prevents small anions from binding to the heine. Our results for the Chromatium protein support this view and indicate that at pH 1 (State B1) the heine is also surrounded by a similar hydrophobic region. The previously observed decrease in the "high spin" 635-nm optical band at physiological pH [14] is qualitatively consistent with our suggestion that the protein at physiological pH corresponds to a quantum mechanical admixture of high and intermediate spin states. The effective paramagnetic moment of R. rubrum ferricytochrome c' at pH 11 has been found to be 6.4/~a at 150 °K and 4.9/~B at 4.2 °K [18]. The former value is about 8 ~ higher than the theoretical high temperature limit of/~erf for a pure high spin ferric heme protein (5.92 #B)- The discrepancy between the experimental and theoretical values of/~eff at 150 °K is most likely spurious, since it is unlikely that

302 there would be such a large orbital contribution to/~erf in the orbital singlet high spin state. The effective paramagnetic moment of protein state Bz measured at 4.2 °K in R. rubrum ferricytochrome c' is only 4 ~ higher than the corresponding low temperature values of #elf listed in Table I for acid metmyoglobin and protein state B2 of Chromatium ferricytochrome c'. The effective paramagnetic moment of ferricytochrome c' at physiological pH has been previously reported to be about 5.15/~, at 293 °K [15], 5.2#B at 150 °K [17] and 3.8 #8 at 4.2 °K [17]; the data taken at 150 and 4.2 °K is for the R. rubrum protein, while the data taken at 293 °K is for the R. rubrum, Chromatium and Rps palustris proteins. The value of #elf obtained for the R. rubrum protein at 4.2 °K is about 1 0 ~ higher than the low temperature value we measured (Table 1) for the Chromatium protein, though still considerably lower than the value of about 4.7 #B usually associated with measurements of #~fr for high spin ferric heme proteins at liquid helium temperatures. The high temperature values of [~eff, 5.16 #~ at 293 °K and 5.2 #B at 150 °K, are both considerably lower than the theoretical high temperature limit for #elf expected for a high spin ferric state. We have made some detailed suggestions concerning the unusual state of the iron ion in ferricytochrome c' at pH 7, but the low susceptibility values alone have led other workers [15] to suggest that the protein at pH 7 exists as a thermal mixture of high and low spin forms, as is commonly found in various ferric hemoglobin and myoglobin compounds [35, 36]. This picture is supported to some degree by the temperature difference optical spectra of the ferricytochromes c' obtained for samples at l0 and 38 °C [15]. The data suggested that in this temperature region the protein is indeed a thermal mixture of high and low spin forms; the optical band at 635 nm commonly associated with high spin iron ions increased as the temperature decreased. Assuming that there is no discontinuity in magnetic properties upon freezing (none were observed), an estimate of the expected low spin population at low temperatures can be obtained from the measurements of/~fr at 150 and 293 °K [37]. The value of~tef f at physiological pH and 293 °K should correspond to 28 ~ of the heroes in a low spin state; at 150 °K the data corresponds to 25 ~o of the heroes in a low spin state. Consequently, we would expect that at 100 °K at least 2 0 ~ of the iron ions should still be in a low spin state. Approximately the same estimate of the low spin population can be obtained from the temperature difference optical absorption spectra and the measurements Of#eft in the high temperature region. The temperature difference optical absorption spectra suggest that the low spin population decreases by no more than about 0.3 ~ between 38 and l0 °C. In conjunction with the high temperature measurements of tUeff, this temperature rate of change of the spin state populations predicts that at 77 °K, 19 ~ of the iron ions should be in a low spin state ; while at 50 °K, 15 ~ of the iron ions should be in low spin state. However, EPR spectroscopy of the ferricytochromes c' at physiological pH, in the temperature range of 100-7 °K, shows no trace of a low spin component. Even allowing for the expected broadening of the low spin EPR signal in the temperature range from 50 to 77 °K, one should be able to detect evidence of the low spin iron ions. For example, magnetic susceptibility measurements on horse erythrocyte catalase [38] are consistent with assuming that at p H 9.0 and 77 °K about 15 ~ of the iron ions of this protein are in a low spin state, and this low spin component of the protein is easily detected by EPR at 77 °K. Thus, there is reason to doubt that the anomalously low values of the para-

303 magnetic moment of the ferricytochromes c' at physiological pH are due substantially to a thermal mixture of high and low spin forms. The more complex alternative, that of a quantum mechanical admixture of ground and excited iron electronic states, appears to be more suitable, since this model can explain the high and low temperature magnetic susceptibility data, as well as the unusual g values of the protein. A preliminary calculation using the spin state admixtures inferred from the g values associated with EPR signal A1, yields a room temperature spin-only value for #eff of about 4.9 #B (to be published). The latter value is only 6 ~ lower than the experimental value previously obtained for the Chromatium protein. The observed temperature-dependent change in the optical band at 635 nm may be due to either line broadening or a thermal mixture of states, involving protein states A1 and A2 and a low spin state; the population of the low spin state would be insufficient to contribute substantially to the paramagnetic moment of the protein. EPR spectra of various ferricytochromes c' at physiological pH and 100 °K have previously been reported [15]. These spectra all show the same general high temperature characteristics which we have observed: an absorption spectrum which is quite broad with the g± peak shifted considerably below g = 6. The variation in the value of g± (from 4.3 to 4.9) in the spectra of the different ferricytochromes c' studied at 100 °K indicates that the spin state admixtures common to the ferricytochromes c' at physiological pH probably vary with the particular species. Further, since one of the ferricytochromes c' previously studied at 100 °K was from Rps. palustris (isolated as a monoheme) it appears that the unique magnetic properties of the ferricytochromes c' are not a result of dimerization. In summary, we have found that in the pH range 1-11 ferricytochrome c' can exist in four protein states, B1, A1, A2 and B2, which can be distinguished by their magnetic properties, and that reversible transitions between these states can be induced by changing the pH of the protein. At physiological pH, in EPR spectral state A, ferricytochrome c' has magnetic properties unlike those of any other known heme protein. The magnetic data are consistent with assuming that at pH 7 ferricytochrome c' can exist in two different protein states, A~ and A2, both of which represent quantum mechanical admixtures of spin states. Protein state A1, the majority protein species at pH 7, consists of an admixture of 65 ~ intermediate spin and 35 ~ high spin.Though thermal mixtures of spin states are quite common in heme proteins, this would be the first example of a quantum mechanical mixture of spin states. At pH 1 (State B1), in the temperature range from 7 to 77 °K, ferricytochrome c' is characterized by magnetic properties which are nearly identical with those of acid metmyoglobin. At pH 11 (State B2) the protein has magnetic properties which are also quite similar to those of acid metmyoglobin; however, acid metmyoglobin has pure axial symmetry at the heme site, whereas in State B2 there is a small rhombic component of symmetry. Ligand binding and organic solvent perturbation experiments support the view that in protein states Ba, Ax and B2 there is a hydrophobic region in the vicinity of the heine which prevents anions from binding to the heine. The term "entatic" or "poised" state, has recently been used to describe a protein state in which the particular conformation of the protein induces a steric or electronic strain at the active site, allowing the protein to be sensitively poised between two different states [39, 40]. Through this mechanism proteins can be thought

304 to acquire a large measure of their specificity a n d versatility. T h o u g h the specific biological f u n c t i o n of cytochrome c' is still largely u n k n o w n , the ability of the oxidized protein to u n d e r g o reversible transitions between four different protein states a n d the u n i q u e magnetic properties of the protein at physiological p H suggest that cytochrome c' m a y be a particularly interesting example of a protein in a poised state. ACKNOWLEDGMENTS We wish to t h a n k Ms Edith A. Shapiro a n d M r Charles Moleski for their valuable assistance in the isolation a n d purification of Chromatium cytochrome c'. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

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