Effect of Metal Substitution on Hemoproteins
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(42) M. J. Potasek and J. J. Hopfield, Proc. Natl. Acad. Sci. U . S . A . , 74, 229 (1977). (43) T. Kihara and J. A. McCray, Bkxhim. Biophys. Acta, 292, 297 (1973). (44) P. L. Dutton, Biochim. Biophys. Acta, 226, 63 (19xx). (45) K. Nakamoto, “Infrared Spectra of Inorganic and Coordination Compounds”, Wiley-Interscience, New York, N.Y., 1970. (46) N. R. Kestner, J. Logan, and J. Jortner, J. Phys. Chem., 78, 2148 (1974). (47) M. F. Perutz, J . Cryst. Growth, 2, 54 (1968). (48) A. G. Redfield, S. D. Kunz, and E. K. Ralph, J . Mag. Reson., 19, 114 (1975). (49) G. Schwarzenbach and J. Heller, Helv. Chim. Acta, 34, 576 (1951).
(68) G. Palmer, G.T. Babcock, and L. E. Vickety, Proc. Natl. Acad. Sci. U . S . A . , 73, 2206 (1976). (69) A. Tzagdoff and M. Lennan, “The Biochemistry of Copper”, J. Peisach, P. Alsen, and W. E., Blumberg, Ed., Academic Press, New York, N.Y., 1966 p 253. (70) D. C. Wharton and M. A. Cusanovich, Biochem. Biophys. Res. Commun.. 37. 1 1 1 (1969). (71) K. G. Brandt, P. G. Parks, 6. H. Czerlinski, and G. P. Hess, J. Bid. Chem., 241, 4180 (1966). (72) F. E. Wood and M. A. Cusanovich, Bioinorg. Chem., 4, 337 (1975). (73) D. 0.Lambeth and G. Palmer, J. Biol. Chem., 248, 6095 (1973). (74) W. G. Miller and M. A. Cusanovich, Bioohvs. Struct. Mech., 1. 97 ,
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(1975). (75) I.M. Kolthoff and W. J. Tomsicek, J . Phys. Chem., 39, 955 (1935). (76) M. D. Llnd and J. L. Hoard, Inorg. Chem., 3, 34 (1964). (77) H. L. Hodges, R. A. Holwerda, and H. 8. Gray, J. Am. Chem. Soc., 96, 3132 (1974). (78) J. V. McArdle, H. B. Gray, C. Creutz, and N. Sutin, J. Am. Chem. Soc., 96, 5737 (1974). (79) L. P. Vernon and M. D. Kamen, Arch. Biochem. Biophys., 44, 298 (1953). (80) S.Taniguchi and M. D. Kamen, Biochim. Biophys. Acta, 96, 395 (1965). (81) K. Dus, K. Sletten, and M. D. Kamen, J. Biol. Chem., 243, 5507 (1968). (82) Q.H. Gibson and F. J. W. Roughton, Proc. R. Soc., London, Ser. 8 , 146, 206 (1957). (83) Q.H. Gibson and F. J. W. Roughton, Proc. R. Soc. London, Ser. B , 147, 44 (1957). (84) T. E. King, prlvate communication. (85) Y.-L. Chlang, L. S. Kamlnsky, and T. E. King, J . Biol. Chem., 251, 29 (1976). (86) E. Stellwagen and R. G. Shulman, J. Mol. Biol., 80, 559 (1973). (87) G. Palmer. Drivate communication. (88i S.Wheriand and H. B. Gray, Proc. Natl. Acad. Sci. U.S.A ., 73, 2950 (1976). (89)J. Rawlings, S.Wherland, and H. B. Gray, J. Am. Chem. Soc., 99, 1968 (1977). (90) 8. I.Swanson and R. R. Ryan, Inorg. Chem., 12, 283 (1973). (91) J. C. W. Chien and C. M. Kao, unpublished results.
6263 (1968). (52) M. S.Tsaoand W. K. WHman~,A&. Chem. Ser., No. 36, 113(1962). (53) J. C. W. Chlen and L. C. Dickinson, J . Bid. Chem., in press. (54) R. Margalit and A. Shejter, Eur. J. Biochem., 32, 500 (1973). (55) R. E. Dickerson and R. Timkovich in “The Enzymes”, 3rd ed, P. D. Boyer, Ed., Academic Press, New York, N.Y., pp 397-547. (56) A. J. Davison and R. T. Hamilton, J . Bid. Chem.,243, 6064 (1968). (57) A. J. Davison, R. T. Hamilton, and L. S. Kaminsky, FEBS Letts., 19, 19 (1971). (58) J. P. Collman, R. R. Gagne, C. A. Reed, T. R. Halbert, G. Lang, and W. T. Robinson, J . Am. Chem. Soc., 97, 1427 (1975). (59) J. M. Rifkind, Biochemistry, 13, 2475 (1974). (60) J. M. Rifkind, Biochim. Biophys. Acta, 273, 30 (1972). (61) T. Tsudzuki and D. F. Wilson, Arch. Biochem. Biophys., 145, 149 (1971). (62) D. F. Wilson, L. G. Lindsay, and E. S. Brocklehurst, Biochim. Biophys. Acta, 256, 277 (1972). (63) R. H. Tiesjema, A. 0. Muijsers, and B. F. vanGeHer, Biochim. B&phys. Acta. 305. 19 (19731. (64) A. 0.‘Muijsers, d. H. Tiesjema, R. W. Henderson, and B. F. vanGelder, Biochim. Biophys. Acta, 267, 216 (1972). (65) J. S. Leigh, Jr., D. F. Wilson, C. S. Owen, and T. E. King, Arch. Biochem. Blophys., 160, 476 (1974). (66) Q. H. Glbson and C. Greenwood, Biochem. J., 86, 541 (1963). (67) G. Greenwood and Q. H. Gibson, J. Biol. Chem., 242 1782 (1967).
Effect of Metal Substitution on the Molecular Structures and Functional Properties of Cytochrome c and Hemoglobin’ James C. W. Chien’,’ Department of Chemistry, Materials Research Laboratory, University of Massachusetts, Amherst, Massachusetts 0 1003, and Max-Planck-Institut fur Biochemie, Munchen. German Federal Republic (Received April 14, 1978) Publication costs assisted by the University of Massachusetts
Metal substitution in hemoprotein provides derivatives for the study of the metal ion as a determinant for the molecular structures and functionalproperties of the proteins and of the protein in governing the coordination chemistry of the metal ion. Comparative studies in our laboratory of the metal-substituted and the native hemoglobins and cytochromes c showed that the protein moiety largely determines the ligands for coordination with the metal ion, its coordination number, and electronic configuration. Metal ion substitution was found to sometimes strongly alter the redox potential and other functional properties of the metalloprotein; the secondary structure of the molecule is not significantly changed in general but some perturbation at the tertiary and quaternary levels have been observed. Introduction Hemoproteins are metalloenzymes comprised of an iron porphyrin prosthetic group and a protein moiety. As a group, the hemoproteins have extraordinarily diverse functions and extremely rich chemistry. Cytochromes are the electron transfer enzymes of life-supporting oxidative phosphorylation exemplifying the fine tuning of redox potentials of the iron porphyrin by the protein moiety. Myoglobins and hemoglobins act as reversible oxygen carriers. Here the globin provides an hydrophobic environment which greatly reduces the propensity of iron porphyrins for a u t ~ x i d a t i o n .Catalases ~ function as cat0022-3654/78/2082-217 l$Ol .OO/O
alysts for chemical reaction with remarkable activities. In the case of hemoglobin, the allosteric transition from one quaternary structure to another gives rise to cooperative function properties and has been much studied as a model for homometallic allosteric enzymes. Tryptophan dioxygenase and cytochrome oxidase are important heterometallic allosteric enzymes. The diverse functions of hemoproteins are certainly the manifestation of the ability of the protein moiety to regulate the coordination chemistry of the iron porphyrin and influence of the latter on the conformation of the protein. The axial ligands play important roles in these 0 1978 American Chemlcal Society
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James
The Journal of Physical Chemistry, Vol. 82, No. 20, 1978
regards: there are two His in cytochrome b5,5one His and ~ one His only in hemoone Met in cytochrome c , ~ -and globin and myoglobin. Ligands can bind to hemoproteins with a stereochemistry which may be quite different or even unprecedented from that for the same ligand in simple iron complexes. For instance, whereas iron carbonyls have uniformly a linear structure except the bridging carbonyl groups, the Fe-C-0 bond angle was found to be 145O by X-ray diffraction for carbonmon. ~the case oxyhemoglobin of Chironomous thumi t h ~ m i In of horse carbonmonoxyhemoglobin, the ligand was found to lie off the heme axis and that the carbon atom may be displaced from the heme axis in the same direction as the oxygen atom.1° The Fe-N-0 angles range from 180" for iron dithiolate complexes to 140" in nitrosyl complexes of tetraphenylporphyrin. By contrast the angle is 110" in nitrosylhemoglobinll and nitrosylhemoglobin.12 In nitrosylhemoglobin of mutant Kansas the subunits are nonequivalent; the Fe-N-0 bond angle is 105O for the 6 subunit and is about 165O for the a subunit. That the posthetic group exerts profound influence on the protein conformation is well recognized. For example, the heme strongly influences the association-dissociation equilibria of hemoglobin. Thus whereas oxyhemoglobin dissociates into unsymmetric dimers upon dilution, deoxyhemoglobin does not; separated apo-a and -6 chains do not associate to form tetramer, Also, the electronic configuration of the Fe atom apparently governs the quaternary and tertiary structures of hemoglobins and myoglobins. The high-spin deoxyhemoglobin and fluoroand aquomethemoglobins are said to have the T quaternary structure, while the low-spin cyan- and azidemethemoglobins and oxy- and carboxyhemoglobins are considered to have the R quaternary structures. The alkaline Bohr effect, the oxidation Bohr effect, and the preferential binding of organic polyphosphates are all consequences of protein conformation changes resulting from ligation and oxidation of Fe. To further elucidate the structure-function relationships between the prosthetic group and the protein moiety, two kinds of studies are particularly instructive. The first one is to focus attention on the variation of the primary structure and to relate it to modifications in functional properties of hemoproteins of different species and mutants, This type of investigation has led to the understanding of clinical anemia at a molecular 1 e ~ e l . lThe ~ second approach is to replace the Fe atom with another metal atom, thus focusing attention on the effect of the metal on the structure and functional properties of the protein molecule. Recent works in our laboratories and elsewhere have demonstrated the feasibility to synthesize a variety of metal-substituted cytochromes c and hemoglobins, The resulting modifications in the physical, chemical, and biochemical properties can often be interpreted by well-known chemical principles. The central purpose of this paper is to show the kinds of biochemical informations obtainable from a comparative study of the native and the metal-substituted proteins.
Synthetic Procedures The choice of a method for metal substitution for a given protein depends upon how the prosthetic group is bound to the apoprotein. Entirely different procedures are used to synthesize metal-substituted hemoglobin and metalsubstituted cytochrome c. The heme b in hemoglobin and myoglobin is held by the apoglobin via van der Waals forces and weak coordinate bond. It has been known for sometimele that the heme can be readily removed by acidification and extracted by
C. W. Chien
ketone. Reconstitution with another metalloprotoheme is rapid and quantitative.16 For best results, the metalloprotoporphyrin IX should be repeatedly purified by silica gel chr~matography,~~ using only an apoglobin preparation which dissolves readily in buffer to give a clear solution, and the metal-substituted hemoglobin or myoglobin purified by cmc chromatography.17 Several metal-substituted hemoglobins have been synthesized but only those of Co and Mn have been thoroughly characterized. Cobalt hemoglobin is the most interesting of all because it binds oxygen reversibly and c o ~ p e r a t i v e l y l ~and - ~ ~exhibits all the allosteric interactions of the native hemoglobin. The heme c in cytochrome c is covalently bonded to the protein via thioether linkages to Cys 14 and Cys 17, so the heme cannot be removed as in hemoglobin. Instead the Fe atom is removed by the following procedure. Native cytochrome c is freed of all moisture in a vacuum line constructed of fluorinated polymer. It is allowed to react with anhydrous HF at 0 O C for 1 min.20 The metal-free porphyrin cytochrome c is immediately separated by G-25 Sephadex chromatography and is used on the same day because of its relative instability. The second departure of synthesis of metal-substituted cytochrome c from that of metal-substituted hemoglobin is the metal insertion reaction. It is well recognized that free transition metal ions destabilize native structure of protein molecules in the approximate order21i22Mn Ni Fe < Co < Cu Zn. Fortunately, the relative rates of incorporation of these divalent metal ions into watersoluble porphyrins follow an opposite order of Cu > Zn > Co, Fe, Mn > Mg, Ni.2s124Even then different procedures have to be developed for insertion of each metal ion which minimizes the concentration of free metal ion and maximizes the rate of insertion, Copper and zinc cytochromes c26 were prepared by simple dialysis of porphyrin cytochrome c against Cu(I1) and Zn(I1) ions at 4 "C, the latter in the dark. Insertion of ions requires optimum concentrations of Co(II), chloride, acetate, hydrogen, and phosphate ions. Nickel(I1) cytochrome c28 was prepared in the presence of glycylglycine and 4 M KCNS, while Mn(I1) insertion2@utilizes 2 M KCNS. Even when the starting metal-free porphyrin cytochrome c is homogeneous, there are sometimes more than one metal insertion products. They are separated by NaCl gradient chromatography on Amberlite CG 50 column discarding those fractions which have electrophoretic and ion-exchange mobilities different from those of the native protein. N
N
N
Effect of Protein on the Prosthetic Group Metal Ion Coordination. There have been many studies on the axial coordination of Lewis base ligands by metalloporphyrin~.~~ Among the metal ions of interest here, copper, zinc, and manganese(II)sl porphyrins prefer to form five-coordinate complexes, whereas manganese(III), cobalt(II), cobalt(III), iron(II), and i r ~ n ( l I Iform ) ~ ~ either 1:l or 1:2 complexes with Lewis base. We are not aware of any report on metalloporphyrin complexation of neutral sulfur containing li ands. In model compound studies, we found that ~ o b a l t ,nickel,28 ~ and copper28protoporphyrin IX dimethyl ester will bind either one nitrogenous base or one thioether molecule but not both at the same time. The results on metal-substituted hemoglobins and cytochromes c described below clearly demonstrate that the protein moiety imposes a particular coordination chemistry on the metal ion. Discussion of the coordination chemistry of hemoproteins centers largely about the coordination number, the identity of the axial ligands, and the extent of dis-
B
Effect of
Metal Substitution on
Hemoproteins
The Journal of Physical Chemistry, Vol. 82, No. 20, 1978 2173
placement of the metal ion from the mean porphyrin plane lengths appears to be a reasonable limited application as as revealed by X-ray diffraction, magnetic resonances, and long as the complexes under comparison involve similar bonding orbitals. The effective ionic radius of high-spin optical s p e c t r o s c ~ p y . ~ ~ - ~ ~ Mn(I1) is 0.82 A as compared to 0.78 A for high-spin Fe(I1) Correlation of visible electronic spectra of metallosuggesting a greater out-of-plane displacement for the porphyrins and metal lop rote in^^^ suggests useful rules for former in porphyrin compounds. The observed distances the determination of coordination number of metal ion in from the metal ion to the mean plane of the four pora given oxidation state and spin multiplicity, Le., low spin phinato nitrogen atoms (M- - -PN) in Mn(1-MeIm)(TPP) or high spin. This is based on the well-documented and Fe(2-MeIm)(TPP)51are 0.52 and 0.42 A,respectively. stereoelectronic shift of the Q and B absorption bands by In deoxyhemoglobin the Fe atom is about 0.75 A out of axial ligands. Based on this criterion, Mn-, Co-, and the heme plane, the Mn atom in M"Hb should be situated Zn-substituted hemoglobins are five-coordinate like the this much or more out-of-plane. As of yet there is no X-ray native deoxyhemoglobin. Likewise, Co-, Ni-, Cu-, and study on MnHb. The structure of Mn(1-MeIm)(TPP) Zn-substituted cytochromes c like the native species are clearly showed51that when the dxzyz orbital is populated, all six-coordinate. The lone exception found in this series the Mn(I1) ion is too far out of the porphyrin plane to is manganese cytochrome c. Therefore, the metal ion permit effective interaction with a sixth (axial) ligand as coordination number in a metalloprotein is largely dewe have proposed to explain the chemical behaviors of termined by the apoprotein moiety. M"cyt c.z9 The effective radii for high-spin Mn(II1) and Axial ligation can also have pronounced effect on the Fe(II1) ions have the same value of 0.65 A.50 A threeredox potential of a metalloprotein. This is a property dimensional difference Fourier map showed the two which measures the ease of removal of an electron from proteins to have very similar structure^.^^^^^ the highest occupied molecular orbital. The redox poThe effective radii for low-spin Co(I1) and Co(II1) ions tential of native high-spin hemoglobin is 0.147 V. The are 0.65 and 0.53 A, respectively." Co(1-MeIm)(TPP)was value is increased to 0.260 V for native low-spin cytofound4zto have M---PN = 0.13 A, similar small out-ofchrome c; its filled d r orbitals lie lower than the d a orplane displacement of the metal atom was also found in bitals. By comparison both five-coordinate cobalt hemother five-coordinate Co(I1) p o r p h y r i n ~ ~and l , ~ ~is also oglobin and six-coordinate cobalt cytochrome c are lowbelieved to be true in CoMb47and CoHb.17319 spin complexes, but the antibonding 3d,z orbital in the latter compound is significantly raised in energy by the Electronic Configurations. Some of the metal substiadditional axial ligand. This is manifested in a sharp tuted hemoproteins are diamagnetic; they are %yt c+, increase in the ease of removal of an electron from cobalt Z n ~ yc,t CoHb+,and &Hb. Others are high-spin complexes: cytochrome c as compared to cobalt hemoglobin; their MnHb,M"cyt c, M"cyt c+, and the model compound for ~~ redox potentials are -0.14 and 0.10 V, r e s p e c t i ~ e l y . ~ ~ *manganese oxyhemoglobin. The remainder have low-spin configurations: C"Hb,CaHbOz,cocyt c, and CUcytc. Manganese cytochrome c is almost certainly five-coordinate. Its Q and B absorption bands occupy almost Single crystal EPR spectra of CoMb47 and CoHb39have identical wavelengths as manganese hemoglobin. The two been previously reported. An analysis of unpaired electron proteins have nearly the same redox potential^,^^ and they density population showed that the electron is found in are both high-spin complexes. The extreme ease of a molecular orbital involving mostly the 3dzzorbital of the autoxidation of manganese cytochrome cZ9and its reaction metal and the 2p orbital of the nitrogen atom of the with nitric oxide are also consonant with the manganese proximal histidine (Table I). The single crystal EPR of ion not being fully coordinated. C"Mb02and its implication of ligand bonding stereochemistry are discussed in a later section. The axial ligands of a metalloprotein can be identified by 'H NMR, EPR, or X-ray diffraction. The 14N suThe EPR spectra of cucyt c showed unambiguously that perhyperfine splitting in the EPR spectra of deoxythe unpaired electron occupies the molecular orbital incobaltomyoglobin47and deoxycobaltohemoglobin39idenvolving the 3d.9. orbital of copper and the atomic orbitals tified His as the axial ligand as it is in the native species.33 of the pyrrole nitrogens. Even though "cyt c is paraX-ray3 and 'H NMR6p8v3"38showed His-18 and Met-80 to magnetic it exhibits no EPR spectrum down to 77 K. be the axial ligands for native cytochrome c. IH NMR and Susceptibility measurementsz8gave an uncorrected value EPR studies44showed these same residues axially coorfor the magnetic moment of 3.7 pug,a value which is high dinated to the metal ion in cobalt cytochrome c. for high-spin octahedral coordinate Ni(I1). This may indicate a considerable amount of zero-field and spin-orbit Perhaps the unique characteristic which distinguishes contribution. The absence of EPR signal could be due to the native hemoproteins from the metal-substituted derivatives is that the spin states and coordination group a zero-field splitting for a distorted octahedral field which geometries of iron(I1) and iron(II1) porphyrins change is larger than the microwave energy. Alternatively, this readily with ligation. In contrast both five- and six-cocould be caused by an extremely short electron spin-lattice ordinate cobalt(I1) and cobalt(II1) porphyrins and proteins relaxation time either because of modulation of zero-field are low spin. Also axial ligands seem incapable of effecting splitting or some Raman mechanism. Even though we did a change of Mn(I1) from a high-spin to a low-spin connot observe any half-field EPR signals, the electronic configuration of "cyt c is probably (3d,z)1(3dX~-yz)1. figuration. MnHb,4' M"cyt c,z9 and M"cyt c+z9 are all high-spin species. Nitric oxide appears to be the only Manganese hemoglobin and cytochrome c are 3d5 ligand, which when allowed to react with manganese(I1) high-spin species. It was not possible to resolve ligand ~ o r p h y r i n sM , ~n~~ ~yc+t~and ~ M n ~ yc,29 t produced low-spin superhyperfine splittings. Single crystal studies are unnitrosyl derivatives. derway in our laboratory. M"Hb and M"cyt c are both The coordination group geometry of the metalloproteins extremely susceptible to autoxidation. However, the should reflect the size of the metal atom. Shannon and dioxygen adduct of Mn(II)(TPP) is stable in toluene at low Prewitt" tabulated effective ionic radii of the elements in t e m p e r a t ~ r e .The ~ ~ authors proposed an Mn(IV)(02)2various oxidation states and coordination numbers. Even valency formalism in which the Mn(1V) is in the 4(t23) though the values are for oxides and fluorides, their use ground state. M n ~ yc+ t has an effective magnetic moment to predict approximate differences in metal nitrogen bond of about 6.45 f 0.6 hB. I t probably has an electronic
2174
The Journal of Physical Chemistty, Vol. 82, No. 20, 1978
James C. W. Chien
TABLE I: Electron Paramagnetic Resonance Spectra and Unpaired Electron Density Distribution of Metal-SubstitutedHemoproteins Cocyt c 2.035 2.223 -61.4 49.8 15.3 12.5
g II
CoHb 2.028 2.326 -79.0 6.0 -17.5 (-10)a
Cucyt c
%yt c
2.216 2.050 183 19.0
2.0 5.73, 6.66 -78
mHb 2.0 5.96 74b
-11.5 -14.5 0.64 0.02 0.30 0.02
0.30 0.10 25
47
I
-712
,
-Y2
I
-3/2
,
-112
112
I
312
512
J
712
M
Figure 1. Variation of line width with nuclear spin quantum number in the electron paramagnetic resonance spectra of single crystal deoxycobaltomyoglobin and oxycobaltomyoglobin.
configuration of (dxy)l(dxz)l(dY~)l(d~z)l; however, one cannot entirely discount (d,) (dxz)(dyz)l(d,Lyz) as a possible configuration. The single crystal EPR spectra of cobalt deoxy- and oxymyoglobin show particularly interesting line-width dependence on the nuclear quantum number, mI,which persists to 4 K. The line width increases with mI in CoMb, but it decreases with the increase of mI in C0Mb02. A possible interpretation is to consider the observed EPR parameters as an average of different tetragonal
distortion^^^
g = (cos2 4 / 2 ) g , a Y z + (sin24 / 2 ) g , z
A = (cos2 r # 1 / 2 ) A , ~+~(sin2 z 4/2)A,z
0.55 0.04
(1)
(2) where 4 gives the direction of the tetragonal distortion. This dynamic Jahn-Teller effect leads to a frequency broadening of the nuclear hyperfine lines AH = d(gll - gJPH + (All - A , h I 2 / h 2 (3) where 7 is the relaxation time for phonon-induced transition between equivalent distortion without reorientation of the electron spin. A plot of the square root'of the EPR line width vs. ml (Figure 1)gave a relaxation time for the 5gConucleus in CoMband CoMb02of 5.65 X and 2.61 x s, respectively. Ligand Binding. Neither protoheme itself nor any of its derivatives binds oxygen reversibly because of rapid autoxidation. Therefore, it is not possible to study the influence of apoglobin on the oxygen binding of native hemoglobin. Model iron porphyrin compounds, which do act as oxygen carrier, cannot be reconstituted with apoglobin.56 In contrast cobalt protoporphyrin IX can reversibly form 1:l adducts with molecular oxygen57thus enabling an investigation of the influence of apoglobin.
29
55
The oxygen affinity of cobalt(I1) protoporphyrin IX dimethyl ester (1-methylimidazole) has been measured from -45 to -31 "C in toluene;58the pl12value calculated at 25 "C is 1.7 X lo4Torr as compared to 5.7 Torr for &Mb. Therefore, the globin moiety increases the oxygen affinity of the prosthetic group by about 300-fold. Both whale and horse skeletal muscle CoMbhave the same free energy changes of 2.4 cal mol-l upon oxygenation, as compared to 5.7 cal mol-l for cobalt p r o t o p ~ r p h y r i n . ~ ~ # ~ ~ The influence of apoglobin from two different species results in substantially different enthalpies and entropies of oxygen binding: for horse the values are A t l = -11.3 cal mol-l, AS = -46 eu; for whale AH = -13.3 cal mol-l, AS = -53 eu. Compared to cobalt porphyrin for which AH = -11.5 cal mol-l and AS = -58 eu, it may be concluded that the enhancement of oxygen binding in horse CoMbis entirely entropic, whereas that in whale CoHbis roughly balanced between enthalpic and entropic components. These results suggest a balance between oxygen-linked changes in the interaction between the prosthetic group and apoglobin, between globin and water, and within the apoglobin. The nature of this balance can vary with apoglobin so that the physiological requirements of hemoglobin of every species can be conserved in the course of evolution. Compared to CoHb,then enthalpy of oxygenation for FeHbis 2-3 cal mol-l of O2 more negative. This accounts for 20- to 50-fold decrease in the oxygen affinity of the cobalt substituted protein.61 In addition, the entropy decrease upon oxygenation is ca. 2 eu larger for FeHbwhich accounts for the remaining difference in their oxygen affinities. Therefore, the lower oxygen affinity of CoHbis largely due to the weaker Co-dioxygen bond than the Fe-dioxygen bond. Kinetic comparison of reversible oxygenation of cobalt and iron myoglobins showed that the two molecules have a comparable rate constant for oxygenation, but the deoxygenation rate constant for C0Mb02 is about 100-fold greater than that for FeMb02. In toluene solutions (-79 "C) dioxygen reversibly binds to M ~ ( T P P ) ( Pwith ~ ) ~replacement ~ of the pyridine to form a five-coordinate complex, rather than by addition to yield a six-coordinate species. This is probably a manifestation of the large ionic radius for the high spin manganese ion. MnHbdoes not bind dioxygen but is instead autoxidized. FeHbhas a strong affinity for CO. The binding of the ligand "antimixes" the 3d,z orbital of Fe with the a orbitals of C0F2 increasing the orbital energy gap between alg (d,z) and eg (dn) to almost 1.5 eV. There is also strong bonding
Effect of Metal Substitution on Hemoproteins
The Journal of Physical Chemistry, Vol. 82, No. 20, 1978 2175
TABLE 11: Helical Content of Metal-Substituted Hemeproteins from Ultraviolet Circular Dichroic Spectra' - __ sample FeHbO, CoHbO, CoHb coHb + IHP "Hb+ % helicity 89.7 87.6 93.1 94.0 87.3 sample cOHb+t IHP coHb+ (pH 8.0) cOHb++ IHP (pH 8.0) 85.5 82.5 % helicity 84.5 sample coHb+ (pH 5.7) coHb+ t IHP (pH 5.7) 85.5 % helicity 87.6 sample %yt c+ Fecyt c Fecyt c+ COcyt c+ % helicity 33.2 28.6 28.6 22.2 crcyt c %yt c sample "cyt c % helicity 28.2 E2.6 24.3 M in IHP, at 25 "C and pH 7.0 unless stated M in metalloporphyrin, 1.0 x a All concentrations are about 1.2 x otherwise.
of the Fe 3da's with the unoccupied la, MO's of CO. In FeHbCO,the alg (d,z) orbital is vacant. In contrast, CoHb does not bind carbon monoxide. This is partly attributable to the populated antibonding alg (d,z) orbital pointing toward the E-helix side of the peptide chain diminishing u bonding interaction and to the fact that the slight out-of-plane position of the Co(I1) ion tends to thwart the back-donating interaction. However, CoTPPhas been reported63to complex carbon monoxide in toluene at low temperatures. Since CoTPPdoes not have a fixed axial nitrogenous base ligand, carbon monoxide can approach the metal ion from the favorable side even if it is displaced out-of-plane. Furthermore, the repulsion of the dZzelectron is reduced by its being able to be more or less pointed toward the opposite axial direction. MnHbdoes not bind CO for similar stereochemical reasons. *"b binds NO strongly and almost irreversibly. The bound NO can only be displaced by photolysis in the presence of C0.64 This probably reflects the stabilization of a back-bonding. M"Hbalso binds NO extremely strong65 and equilibrium studies are not possible. However, NO probably displaces the F8-His to form a five-coordinate complex because of the large ionic radius of manganese (vide supra). In contradistinction, the antibonding d,z orbital is partly populated in CoHb;a backbonding is not a significant stabilizing factor. Thus, CoHbbind NO only weakly and re~ersib1y.l~ F e ~ yctdoes not bind diatomic ligands as is expected and neither does Cocyt c. On the other hand, Mncytc can complex with NO but not with O2 or CO. A plausible explanation is that NO is a good trans ligand and has been shown to promote the severance of the Fe-His F8 bond in hemoglobin Kansas13v66,67 and in HbA when IHP is present.68f69It is likely that the approach of NO to the distal side of the manganese protein labilizes the proximal histidine; this permits another NO to approach the F side to complex with the manganese ion. Interestingly "cyt c reacts readily with NO to give the nitrosyl derivative;28 the paramagnetic complex has EPR parameters of g = 2.187 and g, = 2.140. Under similar conditions N i b ) protoporphyrin IX dimethyl ester in pyridine does not react with NO. Therefore, the protein environment enabled the formation and stabilization of a nitrosyl product. It also implies that the bonds between Ni and the axial ligands in "cyt c is weak and more displaceable than those in Fecyt c and Cocytc. The affinity of FeHb+for various anions is in general not shared by metal-substituted species. Co(II1) and Mn(II1) hemeproteins would have very little if any affinity for anions. This expectation is supported by experimental observations. Since CoHb+only has a weak affinity for the CN- ion, the electronic spectra of CoHb+CN-was stabilized only after many hours. Addition of excess of NS- and Fions has no effect on the spectra. Mncytc+ did not show binding of any anions, whereas MnHb+binds only N, ion weakly.65
Redox Potentials. As important as any other single physical property for hemeprotein is probably the redox potential. It certainly is the thermodynamic determinant for electron transfers; it is also a significant factor in ligand binding since this often involves partial charge transfer. The enthalpy for the reduction of a metalloprotein is the electron affinity of the molecule; the corresponding free energy change is directly related to the half reduction potential.70 The observed redox potentials of various hemoglobin and cytochrome c derivatives can be understood on the basis of the theoretical orbital energies.62 MnHbhas less positive potential (40 mV) than either FeHb (150 mV) or CoHb(100 mV), as can be seen from their orbital energies. That M n ~ yc thas Ern,,= 60 mV which is not much different from that of MnHbis consonant with the fact that they are both high-spin five-coordinate complexes (vide supra). The large difference between the E, of F e ~ yctand &cyt c can be accounted for. Reduction of BecytC+ corresponds to the introduction of an electron into the eg (da) orbital which lies much lower than the a1 (d,z) orbital by about 0.7 V; corresponding reduction o! Cocytc+ would add an electron to the latter antibonding orbital. In the absence of a sixth ligand the alg (d,z) orbital is much lower in energy; in the case of ferrous porphyrin the alg (d,z) orbital for a five-coordinate complex is about 0.53 V lower in energy than six-coordinate complexes.62 All the other metal-substituted hemoproteins have unaccessible redox states as are the corresponding metalloporphyrins. For the latter it is the porphyrin which is electrochemically reduced or oxidized rather than the metal ion. Effect of Metal Ion Substitution on Protein Structures To discern whether a physical perturbation such as ionic strength, and specific anions or a chemical perturbation such as oxidoreduction, ligation, or metal substitution alters the protein structure depends upon the resolution of the technique to detect the changes. Even with high resolution difference Fourier mapping only the most prominent features are described which may have functional relevance whereas numerous smaller differences are either ignored or glossed over. Witness the often encountered assurances that all the liganded hemoglobins have the same quaternary structures. Secondary Structure. Next to the invariant primary structure of a given protein, it can be said that the helix content at the secondary level is not appreciably affected by metal substitution. The helical contents as determined by circular dichroism (Table 11) of metal-substituted hemoglobin and cytochromes c are within experimental accuracy of the native s p e c i e ~ . ~ ~ J ~ Tertiary Structure of Cytochrome c and Conformational Transitions. The conformational structures at the tertiary level are apparently quite similar for the various metal derivatives of cytochrome c. There are, however,
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significant differences in their circular dichroic spectra the details of which will be given e1~ewhere.I~ The lH NMR spectra of cobalt and iron cytochrome c at 300 MHz have been compared.44The resonance positions of the protons of Cys-14, Cys-17, His-18, Trp-59, Tyr-67, Tyr-74, and Phe-82 of both proteins are nearly identical suggesting that these residues of the two molecules have the same spatial positions. The last four residues have been postulated to form an aromatic electron transfer ~ h a n n e l Accord.~~~~~ ingly, cobalt cytochrome c should undergo efficient electron transfer with oxidoreductases. This expectation was not realized. It was found that %yt c is oxidized by cytochrome oxidase at about 45% of the rate for F e ~ yc,27175 t even though the difference in redox potentials between %yt c and cytochrome oxidase is 400 mV more favorable for electron transfer than that between Fecyt c and cytochrome oxidase. Furthermore, Cocytc+ was without activity toward microsomal NADH or NADPH cytochrome c reductase nor by mitochondrial NADH or succinate cytochrome c r e d ~ c t a s e . ~ ~ ~ ' ~ Closer examination of the lH NMR spectra of cobalt and iron cytochromes c offered a plausible explanationfor their different behaviors toward oxidoreductases. There are altogether 19 Lys residues in horse heart cytochrome c. With the possible exception of Lys-5 and Lys-7, the remaining residues are all situated at the surface of the molecule. The protons of these residues in Fecyt c are prominent at 2.95,1.55, and 1.35 ppm.44These resonances are reduced in intensities in Cacytc and cocyt c+. There appeared in the latter cases new resonances of very narrow line widths at 3.00, 1.70, and 1.44 ppm. These may be assigned to those surface lysyl residues of Cocytc have modified structures and are relatively free to rotate; the corresponding residues in Fecyt c are restricted in their motion by interactions such as salt bridges. From the integrated areas of the lH NMR peaks, it was estimated that this modification involves 1-2 lysyl residues in %yt c, and as many as 4-5 lysyl residues in %yt c+. I t has now been firmly established that the binding of both oxidase and reductase to cytochrome c involve some of the surface lysyl residues of the cytochrome c molecule. The binding sites for oxidase and reductase are shown to be different by antibody binding site s t ~ d i e s . ~The ~J~ oxidase binding site probably involve about four lysyl residues, the most important of these are Lys-13 and Lys-22. Chemical modification of the former alone reduces activity by 50%.18 Only 30% of the activity remained when both residues are modified. These may be the same residues which have altered structure in Cocytc. At least eight lysyl residues (5,7,8,72,73,86,87, and 88) have been implicated with reductase binding sites.79 If the lysyl residues in cOcytc+, characterized by shifted and narrowed proton resonance lines, are among the above listed ones, then the molecules's inability of reduction by reductases may be attributed to its inability to bind them. The reduced native cytochrome c has three pH dependent conformational states.71 Metal-substituted cytochromes c all show similar transitions with approximately the same pK values given in parentheses: Mncytc (4.8, 10.9), &cyt c (4.0, 12.0),"cyt c (4.5, 12.0),%yt c (3.2,11.8). It is likely that the transitions result from ionizations of the same peptide residues in these proteins. However, not much more is known about structural changes caused by conformational transitions. EPR studies of the paramagnetic-metal-substituted derivatives revealed the effect of the transitions on the coordination chemistry of the metal ion.
James C. W. Chien
In1
n
Figure 2. Electron paramagnetic resonance spectra of cobaitocytochrome c : (a) pH 8; (b) pH 12.9; (c) pH 0.5.
At physiological pH's Cocytc has an EPR spectrum44 characteristic of a six-coordinate Co(I1) with well-resolved 14N superhyperfine splitting interaction with His-18 (Figure 2a). At pH 1 1 2 , the spectrum (Figure 2b) becomes one of a five-coordinate Co(I1) retaining the 14Nshfs. At pH 1 3 , the unusual spectrum (Figure 2c) can be identifiedso as a five-coordinate Co(I1) having a sulfur containing axial ligand. These results suggest that Met-80 is dissociated at high pH and His-18 is dissociated at low pH. In the case of Mncytc, "cyt c, and %yt c, the five-coordinated metalloporphyrins dimerize at alkaline pH's as evidenced by the characteristic Am = 2 signals at half-field of the triplet species.25t2s>29 For '%yt c, denaturation at acidic pH led further to polymerization of the copper porphyrins. Electronic spectra taken at various pH's for the metal-substituted cytochromes c support the EPR interpretations. Finally, ultraviolet circular dichroic spectra showed that the molecules assumed random-coil configurations at the extreme pH's. Quaternary Structure and Homotropic Interaction. The homotropic interaction for hemoglobin is manifested by the sigmoidal oxygenation curve characterized by a Hill coefficient, n, which is a measure of cooperativity in oxygen binding. The molecule's affinity for oxygen is given by a half-saturation pressure pllz. According to the symmetry conserved model of Monod et al.,sl the homotropic interaction is the result of a quaternary structure change from a low affinity T state to a high affinity R state. Perutzs2 proposed a detailed stereochemical model. For deoxyhemoglobin in the T state the high-spin iron is displaced about 0.75 A out of the heme plane. Oxygenation converts the iron to the low-spin configuration with concomittent movement into the heme plane accompanied by displacement of various peptide residues propagating to the invariant residues at the subunit contacts. In this manner conformational changes initiated by oxygenation of one heme is transmitted through interactions at subunit contact to the other unliganded hemes. One of the reasons for studying the properties of CaHbis to see whether the
The Journal of Physical Chemistry, Vol. 82, No. 20, 1978 2177
Effect of Metal Substitution on Hemoproteins
above triggering mechanism can be applied to CoHbas well. Oxygenation of CoHbis cooperative albeit less than that of native hemoglobin; the Hill coefficient values range from 1.8 to 2.2 for the former,17-19,83@ compared to 2.4-2.8 for the latter. According to Hopfield's distributed energy models4the free energy of cooperation is expected to be proportional to the displacement, 61, of the proximal histidine from the porphyrin plane on going from oxy- to deoxyhemoglobin
AG' =
61(FR - FT)
(4)
where I;k- FT is the difference in forces due to the protein in the R and the T quaternary structures. The values of 61 is about 0.75 and 0.4 A for FeHband CoHb,respectively. The free energy of cooperation is the difference between the free energy of interaction of the first and the last subunits6 to be calculated from the Hill plot by
AG' = AGll - AGJ = 2.303RTfiN
(5)
TABLE 111: Principal Values of Aco and g Tensors for CoMbO, single crystal species I
powdera
gtt gtt g,,
2.081 i: 0.002 1.99 i: 0.01 2.00 It 0.01 A,, 13.1 i: 1.0 A,, 23.5 i: 1.0 A,, 8.8 i: 1.0 species I1 gtt 2.083 i: 0.002 2.084 1.988 1.989 i: 0.002 gss g, 2.006 i: 0.002 2.007 A,, 9.33 r 0.5 10 A,, 16.7 i: 0.5 20 A,, 5.95 i: 0.5 10 a T.Yonetani, H. Yamamoto, and T. Iizuka, J. Biol. Chem., 249, 2168 (1974).
showed that the displacement of the His-F8 and the consequent peptide movement may be the only necessary requirement for allosteric transition. The d,z electron in CoHbrepels the proximal histidine. The Co-N, bond distance is found to be as long as 2.436 A in cOTPP(pip)2. Upon oxygenation of CoHbthe electron is transferred mostly to the dioxygen (vide infra) and the Co-N, bond distance should diminish and become comparable to that in a cobalt(II1) porphyrin complex. It is reported to be 2.060 A in CaTPP+(pip)zand 1.926 A in CoTPPt(Im)2.41 Thus oxygenation of CoHbshould move His-F8 toward the porphyrin plane by 0.38-0.51 8, sufficient to account for its cooperative behaviors. This transfer of electron could also cause a transition from an initially nonplanar porphyrin core to essential planarity on oxygenation of &Hba41 In conclusion, the only necessary and sufficient condition for allosteric transition of hemoglobin in the movement of the His-F8 upon oxygenation. The oxidation of MnHbis also found to be cooperativeae6 The redox n values, with and without organic phosphates, are similar to those of FeHbat all pH values.@3
where N is the normal distance separating the asymptotes at very high and very low degrees of oxygenation. The free energy of cooperation is 1448 cal mol-l for cobaltohemoglobinas and 4590 cal mol-l for ferrohemoglobin at physiological pH. The former is about one-third of the latter. Whereas, the ratio of 61 of CoHbto FeHbis about 0.5. Therefore, the forces due to the protein is smaller in CoHbthan in FeHb according to the distributed energy model. In terms of the allosteric modele1 the weakened subunit interaction in CaHbmay be interpreted as a result of either a shift of the equilibrium toward the quaternary R structure or a somewhat relaxed T structure. The two interpretations above both find experimental support. From the oxygenation equilibrium curves the allosteric constant, which is the ratio of the population of the T state to that of the R state in the absence of oxygen and 4,8 according to the allosteric model,81is 5.2 X X lom2 for FeHband CaHb,respectively. Therefore there is a shift of equilibrium to the quaternary R structure for Quaternary Structure and Heterotropic Interactions. CaHb. CoHbwas found to release the same number of Bohr That the T structure for CoHbis more relaxed than the protons upon oxygenation as FeHb88p88 at low ionic strength T structure of "Hb is found by circular dichroism studies.17 in the absence of chloride ions. However, differential The changes in CD and UV spectra at 287 nm have been titration in the presence of chloride ions titrate only a proposed to arise from changes in the environment for fraction of the Bohr protons; it is about 60% in 0,l M Tyr(C2)P and Tyr(C7)a situated at the CUPcontacts and NaC1,8s and only 40% in 0.3 M NaCl.lg This can be intherefore can be utilized as quaternary i n d i c a t ~ r . ~ ~ - @terpreted ~ to mean that the salt bridges in CoHbare weak Spectra in this region for CaHband CaHb02showed that and sensitive to dissociation by chloride ions. In other there is definitely a shift in the quaternary structure with words, CoHbhas a more relaxed T structure than the native ligation, The changes in spectral features are very similar protein. to those accompanying ligation of the native proteins. Organic polyphosphates lowers the p1lzof FeHb and However, the changes are smaller for the cobalt derivatives. CoHb by the same degree.l@le3 In terms of Adair The differences in ellipticity, A[8], = [8];w - [elydeoxy, is constant^^^^^^ 2,3-diphosphoglycerate lowers the overall greater for the native species: A[8]287= 2.0 X lo4 and oxygen affinity and increases the cooperativity by reducing A[8]2@l= 1,4 X lo4. The corresponding values are A[8]28, kl,k2,and k3 without causing a significant change in kl. = 1.07 X lo4 and A[8]zgl= 0.44 X lo4 for cobalt hemoOn the other hand, inositol hexaphosphate lowers the globin. If these A[& values reflect the extent of quaoverall affinity by reducing all k values. ternary conformational changes, then they should be Stereochemistry of the Metal-Dioxygen Bond related to the free energy difference, AG', between the R and T quaternary structures. Literature on native Detailed stereo- and electronic structures of the cohemoglobins and the above results for cobalt hemoglobins balt-dioxygen bond in oxycobaltmyoglobin have been seem to support such a correlation. determined by single crystal EPRS4' The spectral parameters are given in Table 111. The following conclusions The results for CoHbsuggest the following modification for the triggering mechanism for allosteric transition. Since can be made from the data. First, the principal g values ion trapped in both CaHb and C0Hb02have low spin configurations, are very close to those found for the 02mat rice^^^^^^ with principal g values of 2.088, 2.008, and changes of spin-state postulated by Perutz is not necessary. 2.000. The unpaired electron in CoMbOzmay be said to Also the cobalt atom in both CaHb and CoHb02is not be primarily localized on the dioxygen ligand.es Therefore, appreciably situated out of the porphyrin plane; movement the g tensor principal axes defines the orientation of the of the metal atom as postulated by Perutz is also not dioxygen. Secondly, the g and CoAtensors do not share essential for allosteric transition. The results with CoHb
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James C. W. Chien
Fine controls of Eo in cobalt analogues of the hemeproteins would not be possible. CoHbhas Eo only 50 mV more negative than FeHb. However, a second axial ligand raises the antibonding alg orbital greatly; Cocytc has Eo 400 mV more negative than Fecytc. This orbital is also orthogonal to the porphyrin a orbitals. Thus, substituents on the porphyrin ring will have minimal influence on EO. I t is reasonable to expect that all six-coordinate cobalt hemeproteins would have very negative Eo and five-coordinate species have Eo 100 mV. There will probably be even less variation of Eo's among manganese analogues. Because of the metal ion's out-of-plane position in both oxidation states, axial ligands would have little effect on the Eo of manganese hemeproteins. It appears perfectly clear that iron porphyrins are the only prosthetic groups that nature can select to execute the biological functions provided by hemeproteins.
-
Flgure 3. Principal axes systems for the COA tensor ( x , y , z ) and the g tensor (t,11, 0 of oxycobaltomyoglobin.
the same principal axes systems; their relationships are shown in Figure 3 where the coordinates ( x , y, z ) and (4, 7, are for CoAand g tensors, respectively. From this it is seen that the dioxygens are a bonded to the cobalt nucleus in &Mb02. Finally, there are two distinct dioxygen adducts with the same g values but differ in their CoA values (Table 111). The projections of their 0-0 axis onto the porphyrin plane are related by a 90" rotation. The EPR structure makes molecular orbital senses. The j' and 7 axes define, respectively, the planes for the ax*and ay*orbitals of dioxygen. The reason that these are rotated 35" about the 5 axes is to permit overlap of both a* orbitals with the d,, and dyzorbitals for one of the species (I). The a-orbital overlaps for the second species (11) is half in-phase and half out-of-phase, suggesting a slight tilting of the 0-0 axes with respect to the heme plane (the accuracy of EPR measurements permits this angle of tilt to be up to 10"). This reduces the electron transfer from Co to 02. It is likely that the existence of two different kinds of bound dioxygen is also influenced by the trans effect of His-F8. For species 11,the a* orbital of imidazole overlaps in phase with the d r orbital of Co, and the trans effect weakens the binding of dioxygen and reduces the amount of electron transferred to it. These orbitals overlap out-of-phase for species I. This interpretation implies different unpaired electron densities on the dioxygen and the cobalt ion of the two species.
r)
Biological Significance The study of metal substituted hemeprotein not only permit the experimentations and understanding of structure-function relationships at specific molecular levels, it also provides a deep appreciation of the biological uniqueness of iron porphyrins. Porphyrins are among the most stable organic molecules; its presence is detected even in interstellar space. Given porphyrins as a building block material for hemeproteins, then only the Fe derivative can provide the varied and finely controlled structures and functions needed by living organisms. This is due largely to the fact that depending upon its spin multiplicity and oxidation state Fe can assume either an in-plane or outof-plane geometry, and that in the low-spin states Fe is devoid of antibonding electrons. Take the electron transfer enzymes of the oxidative phosphorylation sequence; for example, the Eo's are 60, 220,240,290, and 550 mV for cytochromes b, cl, c, a, and u3, respectively. It is about -410 mV for cytochrome P450 and 150 mV for FeHb. Thus by varying the sixth ligand which is RS-, His, Met, and none, in cytochromes P450,b, c, and FeHb, respectively, a continuous range of Eo is available. Furthermore, the d a orbital of an in-plane Fe overlaps strongly with p r orbitals of the porphyrin; ring substituents can have significant effect on Eo. For instance a formyl group on position 8 makes cytochrome u3 strongly oxidizing.
References and Notes This work was supported in part by the National Institute of Heart and Lung Grant HL-14270 of the U.S. Public Health Service, by the Materials Research Laboratory of the University of Massachusetts, by the Alexander von Humbolt Stiftung, and by the Max-Planck-Institut fur Biochemle in Muchen. Dedicated to Professor John E. Willard of the Universliy of Wisconsin on the occasion of his 70th birthday. Abbreviations used are; CD, circular dichroism; Fscyt c , lron(I1) Fen ~ yc+, t Fe(II1) cy! c ; %yt c, manganese(I1) cytochrome c ; M cytochrome c; cyt c , manganese(II1) cytochrome c ; &cyt c, cobalt(I1) cytochrome c; %yt c', cobalt(II1) cytochrome c; "cyt c, nickel(I1) cytochrome c; %yt c , copper(I1) cytochrome c ; %yt c, zinc(I1) cytochrome c; DPG, 2,3diphosphoglycerate; L?, reduction potential; EPR, electron paramagnetic resonance; g, tensor; A, hyperfine tensor; FeHb, iron(I1) deoxyhemoglobin; eHb+, methemoglobin: FeHb02,iron(I1) oxyhemoglobin; &Hb, cobalt(I1) deoxyhemoglobin; &Hb+, cobalt(II1) hemoglobin; &Hb02, cobalt(I1) oxyhemoglobin; M"Hb, manganese(I1) deoxyhemoglobin; MnHb+, manganese(II1) hemoglobin; Im, imidazole; IHP, inositol hexahosphate: M, methylene blue; "Mb, cobalt(I1) deoxymyoglobin; "Mb02, cobalt(I1) oxymyoglobin; pip, piperidine; 'H NMR, proton magnetic resonance; p i12, half oxygen saturation pressure; [B],, molar ellipticity. J. H. Wang, A. Nakahara, and E. B. Fleischer, J. Am. Chem. Soc., 80, 1109 (1958). J. Ozoles and P. Strittmatter, J. Bid. Chem., 239, 1018 (1964); 241, 4787 (1966). C. C. McDonald, W. D. Phillips, and S. N. Vlnegradov, Biochem. Biophys. Res. Commun., 36, 442 (1969). R. K. Gupta and A. G. Redfield, Science, 169, 1204 (1970). C. C. McDonald and W. D. Phillips, Biochemistry, 12, 3170 (1973). R. Huber, 0. Epp, and H. Formanek, J . Mol. Biol., 52, 349 (1970). E. J. Heidner, R. C. Ladner, and M. F. Perutz, J. Mol. Biol., 104, 707 (1976). J. C. W. Chlen, J . Chem. Phys., 51, 422 (1969). L. C. Dickinson and J. C. W. Chien, J . Am. Chem. Soc., 93, 5036 (1971). J. C. W. Chien and L. C. Dickinson, J. BbL Chem., 252, 1331 (1977). M. F. Perutz and H. Lehmann, Nature (London), 219, 902 (1968). M. L. Anson, and A. E. Mirsky, J. Gen. Physiol., 13, 469 (1930). A. Rossi-Faneili, E. Antoninl, and A. Caputo, Biochim. Biophys. Acta, 30, 608 (1958). J. C. W. Chien and F. W. Snyder, Jr., J . Biol. Chem., 251, 1670 (1976). B. M. Hoffman and D. H. Petering, Proc. Nafl. Acad. Sci. U.S.A ., 67, 637 (1970). L. C. Dickinson and J. C. W. Chien, J. Bbl. Chem.,245, 5005 (1973). A. B. Robinson and M. D. Kamen, "Structure and Functions of Cytochromes", K. Okinuki, M. D. Kamen, arid I.Sekuzu, Ed., University Park Press, Baltimore, Md., 1968, p 383. P. H. von Hippel and T. Schleich, "Structure and Stability of Biological Macromolecules", S. N. Timasheff and G. D. Fasman, Ed., Marcel Dekker, New York, N.Y., 1969. E. P. Chang and J. C. W. Chien, Biopo/ymers, 12, 1063 (1973). E. I. Choi and E. B. Fleischer, Inorg. Chem., 2, 94 (1963). T. P. Stein and R. A. Plane, J. Am. Chem. Soc., 88, 2525 (1966). M. Findby, L. C. Dickinson, and J. C. W. Chlen, J. Am. Chem. Soc., 99, 5168 (1977). L. C. Dickinson and J. C. W. Chien, Biochem. Biophys. Res. Commun., 58, 236 (1974). L. C. Dickinson and J. C. W. Chien, Biochemistry, 14,3526 (1975). M. Findlay and J. C. W. Chien, Eur. J . Biochem., 76, 79 (1977). L. C. Dickinson and J. C. W. Chien, J. Bbl. Chem., 252, 6156 (1977). J. W. Buchler, "Porphyrins and Metalloporphyrlns", K. M. Smith, Ed., Elsevier, New York, N.Y., 1975, pp 207-210. r:
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