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Electrochemical studies of porphyrin redox reactions as cytochrome models
Bioelecirochemistry
and
Bioenergefics
I,
r 72-r
Electrochemical as Cytochrome
Studies of Porphyrin Models *
79
(1974)
Redox Reactions
by GEORGE S. WILSON Department
of Chemistry
University
of Arizona
Tucson,
235721 U.S.A.
A series of Fe(II1) derivatives of mesoporphyrin IX have been These derivatives contain histidine and esamined electrochemically. methionine which have been covalently attached to the porphyrin ring vin the propionic acid side chains. These studies show that the attached ligands are capable of intramolecular coordination with the iron to give distinctive electrochemical properties. The pH-dependence of the formal potentials for the various derivatives is presented in the light of the
characteristics of cytochrome compounds for electrochemical
-
c. Various approaches studies are reviewed.
to heme model
--
The prevalence of heme proteins in biological electron transport systems has stimulated considerable study of their physical and chemical properties. The recent resolution 1 of the X-ray structure of the reduced form of cytochrome c combined with that of the oxidized form provides some important guidelines for the study of cytochrome electron transfer. For example, the inaccessibility of the z-axis ligands (histidine-IS and methionine-So) coordinated with the Fe- protoporphyrin IX raises important questions concerning their role in electron transfer to the central iron atom from a substrate_ Further, if regions of accessibility to the heme group from the exterior of the protein define possible reaction sites then the edge of the porphyrin ring in the oxidized form constitutes a point of substrate attack_ The understanding of these complicated processes will undoubtedly be enhanced by the development of meaningful model systems to which the native protein can be compared. The simplest model systems (at least in terms of nominal structural complexity) are those involving complexes of Fe- protoporphyrin IX with exogenous ligands such as nitrogen bases. The foundation for this work is the elegant potentiometric studies of CL_~RK and co-workers. 2
Pont
* Presented at the 2nd International B Mousson i-5 Oct. 1973
Symposium
on Bioelectrochemistry
Electrochemical Studies of Porphyrin Redox Reactions
173
More recently, polarographic and cyclic voltammetric studies of the reduction of iron porphyrin complexes have provided additional details concerning the solution equilibria involved. a-5 Unfortunately, studies of these systems in aqueous solution are complicated by extensive .aggregation which is due to the large size and lack of hydrophilic groups on the porphyrin molecule. 6 This situation leads to intermolecular associations ranging from distinct dimeric species 7$ to aggregates corresponding to hexamers or even higher order polymers. 9 Dissociation of these aggregates can occur at such rates that kinetically-controlled polarographic waves can be observed. s These effects can be minimized by working with dilute solutions (IO-~ M or less), mixed ,solvents or by addition of strongly coordinating esogeneous ligands. Such measures are necessary in order to assure that reaction kinetics being measured are not dominated by aggregation phenomena. -While aggregation is an intrinsic property of these “ simple ” model systems, great care should be taken in attributing any biological significance to these interactions. Studies of these compounds have shown, however, that the z-axis ligand has a significant effect on the thermodynamics of electron transfer ‘and there is also some evidence that modification of the Fe spin state by the ligand can moderate the rate of electron transfer as welLlo Because the Fe-porphyrin (heme) moiety is covalently bound to the peptide chain in cytochrome G, it is possible to isolate .this section of the protein by pepsin, chymotrypsin and/or trypsin-catalyzed hydrolysis of this protein. A second type of model compound, the heme peptide, can be obtained. The amino acid sequence around the point of covalent attachment is shown below.
val-gln-lys-c~s-ala-gln-c~s-his-thr-val-~l~-l~s-~l~-~l~-l~~~s II
I I( I-fI(
I2
13 1 I4
15
16
17
18
rg
zo
21 I22
-+I
k-y----HP8 HP1r
_ --+I HP15 HP16
23
1
24
251 261
I I
I I
PI’
I>
PI
In a series of potentiometric measurements, HARBURY and LOACH~J” used the pH dependence of the formal potentials for the one-electron reduction of heme peptides HP8 and HPrr to study z-axis ligand interactions. Over the pH range 4-12 several species might exist as ligands : OH-, H,O, .c-amino(lysine), imidazole (histidine), the heme ,.ring propionate group, and the a amino group of the N-terminal residue. Analogous exogenous ligands were also added. By comparison of apparent proton-linked functions with known dissociation constants for. amino acid functional groups, ligand assignments- in the various pH regions
Wilson
174
studies were carried out spectrophotometrically by and HP16. In general, histidine emerges as the most probable ligand in the physiological pH range. Evidence exists I3 for the involvement in HPII and HP15 of the a-amino groups of nterminal valine-11 in this pH range. At higher pH the c-amino groups can be involved. A comparison of the l&and binding characteristics of free imidazole and that of histidine-I8 indicates clearly that the formation of a macrocyclic ring (porphyrin plus peptide chain) makes the histidine a significantly better ligand than free imidazoleIn fact, histidine-I8 is probably a ligand at pH 4.2. Is Histidine-a6 behaves more like free were made.
Similar
\VAP,ME 1%for HP15
imidazole as expected. We have examined the electrochemistry of the above compounds at the DME and pyrolytic graphite electrodes. g Unfortunately (vi& SZ@Z) these techniques are especially vulnerable to inIIuence by aggregation effects. II-l3 All of the heme peptides are electroactive but, with the exception of HP16, give long drawn out waves. The presence of strongly coordinating ligands (histidine-I8 and -26 in HP16) or imidazole added to HPrr or HP15 tends to break up aggregation and yield polarographic waves of reasonable shape. Under these conditions all of the derivatives exhibit essentially the same electrochemical and spectral behavior_ The observed half-wave potentials were within IO mV of the potentiometric value for HP16 (see Tab. I) and no significant differences in the kinetic behavior were noted.
Table I.
Formal potentials for heme models
Compounds
1 W”
(pH 7.0)~ V
Medium
Reference
+0.256
Phosphate
I4
-0.233 -0.23 -0.19 -0.207
0.05 M Phosphate 0.05 M Phosphate -
I3 I3 =5
-0.220 -0.221 -0.210 -0.074 -0-05
b imidazole buffer b b 0.05 M Phosphate/ 0.05 M Acetate b b
! I.
2. 3_ 4. 5. 6. 7_ 5. g_
IO.
Cytochrome
c
(horse heart) HP16 HPI~ HPII HP8 Bis-his-mesoheme HP8 + imidazole Monohis-mesoheme Hismet-mesoheme HPP + n-acetyl
dZ-methionine I I. Monomet-mesoheme
12. Bis-met-mesoheme a Potentials
4.004 +0.020
given with respect
(see Experimental) b Medium -0.1
KH2P0,/K2HP0,
to the standard
hydrogen
M Phospate buffer pH 7.0 30 o/o V/V
12 16 II 16
this work I7 .
electrode
16 16 (SHE)
dioxane-water
Electrochemical
Studies
of Porphyrin
Redox
Reactions
=75
In the present work we will describe an approach t-o porphyrin model compounds for electrochemical studies in which the amino acids of interest are covalently bound to the porphyrin ring. 1*-20 The advantage of this approach over the heme peptides is that the number of possible ligands can be restricted while still maintaining a covalent linkage. Further, the approach makes possible the examination of methionine as a ligand since it is now known to be an axial &and in both oxidized and reduced cytochrome c. 1
Experimental Apparatzrs
and methods
Cyclic voltammetric data were obtained using the apparatus described previously. 21 Polarographic measurements were made at 25 OC in an H-cell constructed from a pyres cuvette which required about 1.5 cm3 of solution. Spectral measurements could also be made in this cell if desired. _4n Ag IA&l reference electrode was used for these measurementswhich had a potential of +o.zoo V ZJUS. standard hydrogen electrode (SHE). The pH measurements were made using a SARGENT miniature combination electrode standardized against NBS buffers. The apparent pH measurements in the dioxane-water mixtures were made on this basis. Materials
The synthesis of the mesoheme derivatives is described elsewhere. Is Stock solutions of these compounds were prepared in potassium phosphate buffer whose pH was adjusted with potassium hydroxide. The 30 yO (V/V) dioxane-water solutions were prepared from freshly distilled dioxane, 22 and stored in the dark. This is quite important because the peroxide impurities are both electroactive and destructive to the mesoheme derivatives .
Results and discussion Polarograplty
of mesoheme
derivatives
.
Fig. I shows the structure of one class of mesoheme derivatives Z-methionine is covalently attached to the porphyrin ring studied. through the carboxyl group of the propionic acid side chain. If the substi,tuent, R attached to the other side chain is Z-histidine, then a histidinemethionine (his-met) derivative results. rs In this way the various mesoheme compounds shown in Table I are obtained. The electrochemical experiments described below were carried out under conditions carefully defined by the rather detailed spectral studies described elsewhere. 16
y2
CH2 I
72
C”, I
Fig. I. Structure of a methionine R = histidine, methionine.
COR
CoNHiHco27”2
- mesoheme derivative. A.B = C,H,.
y2 7 C”3
The pH range (6.5-11.0) over which the measurements were made was defined by the existence of reversible one-electron waves with diffusion controlled limiting current. The pH dependence of the bis-his-derivative is shpwn in Fig. 2. The observed behavior is very similar to that obtained by HARBURY and LOACH u for HPS with added imidazole (Table I). The upward break in the curve at an apparent pH of 7 .5 is characteristic of a proton-linked function associated with the ferroheme form. In view of the fact that spectral evidence strongly suggests the esistence of a bis-histidine complex at this pH, the pH dependence reflects the protonation of the imidazole ring. Since the pK for histidine is 6.1 then there is either some steric hincirance to comples formation or alternatively the interaction of another ligand. _4t about pH II there appears to be another break in the cxve which is characteristic of an acid-base reaction involving the osidized form and probably histidine. The curve for the monohistidine case is characteristic of a high Spectral evidence suggests that the histidine is strongly spin svstem. coordinated in the oxidized and reduced forms. It should not be assumed, however, that the convergence of the mono- and bis-histidine potentials at pH 6.5 means that we are dealing with the same process. In the latter case reduction clearly occurs without change of ligands while in the former water and/or hydroxide may be involved. Preferential stabilization of the Fe(I1) o-xidation state is indicated by the dramatic shift to more positive potentials exhibited by the his-met-, mono-met- and bis-met-derivatives {see Table I>. The behavior of his-metis of particular interest because it possesses the two Iigands found in cytochrome c_ The potentiaLpI curve for the latter resembles the bis-his-
Electrochemical
Studies
of Porphyrin
Redox
Reactions
177
curve in terms of shape although it is shifted about 3.5 p_H units lower c which and over 450 IEV more positive. I4 In contrast to cytochrome exhibits low spin spectra at pH 7 as does bis-his-, the spectrum of the his-met-derivative has characteristics of both high and low spin, possibly mixed. 16 The potential of his-met at pH 7 is in reasonable agreement with that of compound IO (Table I). Above pH 7 the shape of the hismet. monomet and bismet curves reflect the decreasing ability of methionine to form a strong complex with the ferri-heme. The l&and replacing
l-E
-50
-
-100
-
-200
‘7
-250
-
I
6.0
1
I
I
I
I
7.0
8.0
9.0
10.0
11.0 PH
Fig. z. Formal (a
dependence of some mesoheme derivatives. in 30 010 (V/v) dioxane-water mixture. (A) monomet, (e) hismet, (e) monohis, (A) bishis.
potential
approximately
bismet.
2
-
X
pH
10 --1 M
Heme
concentration
Wilson
178
the methionine would be either water and/or hydroAxide. KOLSKI and PLANE ~3 have shown that the reaction H,O-FeP-H,O
+ H+ + H,O-FeP-OH
(I)
where FeP stands for the Fe(II1) derivative of deuteroporphyrin 2,4disulfonic acid [Fe(DSS)] has a pK of 7.7. The divergence of the monomet and bismet cuxres below pH 7 suggest s the increased involvement of both metbionine ligands in complex formation.
Conclllsions
It is clear from this study that the introduction of methionine as a well defined interacting ligand in place of histidine results in a positive shift in formal potential which is indicative of increased stabilization of the lower iron oxidation state. Water as a ligand also causes a positive shift which is not as great as that of met’hionine. Favorable conditions for iron methionine interactions require the formation of an Fe(U) methionine complex at a pH somewhat less than 7 where other potential ligands cannot compete effectively because they are no longer basic in nature. However, in spite of the introduction of methionine, the formal potentials for these model compounds differ greatly from cytochrome c. Such model systems apparently do not approximate the heme environment nor do they duplicate the role of the peptide chain in orienting the methionine properly. The recent elegant reconstitution studies of cyanogen bromidecleaved cytochrome c by CoRR.ADIx and HARBURY 34have suggested the possibility of further study of cytochrome c models which more fully approximate the behavior of the native protein.
Acknowledgments
We would like to thank L. P. HAGER and P. IL WARME for the generous gift of the mesoheme derivatives. This work was supported in part by National Science Foundation Grant GP-28051.
References 1
T. TXKXNO, R_ SWANSON, Harbor
2
W.&f.
O_B_ KALLAI and R.E. DICKERSON, Cold Spring Symposium on Quantitative Biology, 36, 397 (1971) CLARK, Oxidation-Reduction Polenfiak of Organic Systems, Williams
and WiMns,
Baitimore (1960)
Electrochemical Studies of Porphyrin Redox
Read&s
.....-.,’
,. 174
DAVIS ahd R.F. MARTIN. J. Amer. Ci?em. SOL 86, 1365 (1966) BEDNARSKI and J. JORDAN, J. Amer. Chem. Sot. 89,.x552 (1967) DAVIS and J.G. MONTALVO, Anal. Chem.;,,41,_ 1195 (rg6g) _:’ : ;;: c.I:,.. FALK, Pwphyrins and lkfetaZZopwphy&, Elsevi&, Amsterdam, New (1964) 7 FLEISCHER, J.M. PALMER, T.S. SRNASTAVA and.A. CHATTE&J~E,J. .~ Amer. Chem. Sot. 93, 3162 (1971) 8 G.S. WILSON and BP. NERI, AM. N. Y. Acad. Sci. 206,, 568. (1973) ,: 9 G.S. WILSON, unpublished results 10 KM. KADISH and D.G. DAVIS, Ann. N.Y. Acad. Sci. in press ll H.A. HARBURY and P.A. LOACH, J. BioZ. Chem. 235, 3640 (1960) 1s H.A. HARBURY and P.A. LOACH, J. Biol. Chem. 235, 3646 (1960) 13 P.K. WARME. Ph. D. Thesis. University of Illinois, Urbana (1969) 14 -_. ’ F.L. RODKEY and E.G. BALL, J. BioZ. Chem. 182, 17 (1950) 15 H.A. HARBURY,and P.A. LOACH, Proc.Naf. Acad.Sci. U.S.e45,13&(rg5g) 16 P.K. WARME and L.P. HAGER, Biochemistry 9,‘1606 (Ig7b) .17 H.A. HARBURY et a$.,Proc. Nat. Acad. Sci. U.S.: 54, 1658 (1965) 16 W. LAUTSCH, R. PASEDAG. I. SOMMER, H.J. JULIUS and E; BOEDERFELD; Ctrimia 13, 129 (1959) 19 P.K. WARME and L.P. HAGER, Biochemistry 9, 1599 (1970) 20 A. VUI DER HEIJDEN.H.G.PEER~~~A.H.A. VAN DEN OORD. J_Chem. Sot. D.G. T.M. D.G. J.E. York E.B.
Chem.
Gomm.
1971,
369
c : .: B.P. NERI and G.S. WILSON, Anal. Chem. 44, ioo2 (1972). '. L.F. FIESER, Experiments in Organic Chemistry, D.C. HeathCo:.(Igqr)p.36g _: G.B. KorsRr and R.A. PLANE, Ann. N-Y. Acad. Sk., in press : G.CORRAVIN~~~H.A. HARBURY, Prw.N& Acad. Sci..U.S;68,3036(x471)’
. L
k i,
?
:.
and
Bioenergefics
I,
r 72-r
Electrochemical as Cytochrome
Studies of Porphyrin Models *
79
(1974)
Redox Reactions
by GEORGE S. WILSON Department
of Chemistry
University
of Arizona
Tucson,
235721 U.S.A.
A series of Fe(II1) derivatives of mesoporphyrin IX have been These derivatives contain histidine and esamined electrochemically. methionine which have been covalently attached to the porphyrin ring vin the propionic acid side chains. These studies show that the attached ligands are capable of intramolecular coordination with the iron to give distinctive electrochemical properties. The pH-dependence of the formal potentials for the various derivatives is presented in the light of the
characteristics of cytochrome compounds for electrochemical
-
c. Various approaches studies are reviewed.
to heme model
--
The prevalence of heme proteins in biological electron transport systems has stimulated considerable study of their physical and chemical properties. The recent resolution 1 of the X-ray structure of the reduced form of cytochrome c combined with that of the oxidized form provides some important guidelines for the study of cytochrome electron transfer. For example, the inaccessibility of the z-axis ligands (histidine-IS and methionine-So) coordinated with the Fe- protoporphyrin IX raises important questions concerning their role in electron transfer to the central iron atom from a substrate_ Further, if regions of accessibility to the heme group from the exterior of the protein define possible reaction sites then the edge of the porphyrin ring in the oxidized form constitutes a point of substrate attack_ The understanding of these complicated processes will undoubtedly be enhanced by the development of meaningful model systems to which the native protein can be compared. The simplest model systems (at least in terms of nominal structural complexity) are those involving complexes of Fe- protoporphyrin IX with exogenous ligands such as nitrogen bases. The foundation for this work is the elegant potentiometric studies of CL_~RK and co-workers. 2
Pont
* Presented at the 2nd International B Mousson i-5 Oct. 1973
Symposium
on Bioelectrochemistry
Electrochemical Studies of Porphyrin Redox Reactions
173
More recently, polarographic and cyclic voltammetric studies of the reduction of iron porphyrin complexes have provided additional details concerning the solution equilibria involved. a-5 Unfortunately, studies of these systems in aqueous solution are complicated by extensive .aggregation which is due to the large size and lack of hydrophilic groups on the porphyrin molecule. 6 This situation leads to intermolecular associations ranging from distinct dimeric species 7$ to aggregates corresponding to hexamers or even higher order polymers. 9 Dissociation of these aggregates can occur at such rates that kinetically-controlled polarographic waves can be observed. s These effects can be minimized by working with dilute solutions (IO-~ M or less), mixed ,solvents or by addition of strongly coordinating esogeneous ligands. Such measures are necessary in order to assure that reaction kinetics being measured are not dominated by aggregation phenomena. -While aggregation is an intrinsic property of these “ simple ” model systems, great care should be taken in attributing any biological significance to these interactions. Studies of these compounds have shown, however, that the z-axis ligand has a significant effect on the thermodynamics of electron transfer ‘and there is also some evidence that modification of the Fe spin state by the ligand can moderate the rate of electron transfer as welLlo Because the Fe-porphyrin (heme) moiety is covalently bound to the peptide chain in cytochrome G, it is possible to isolate .this section of the protein by pepsin, chymotrypsin and/or trypsin-catalyzed hydrolysis of this protein. A second type of model compound, the heme peptide, can be obtained. The amino acid sequence around the point of covalent attachment is shown below.
val-gln-lys-c~s-ala-gln-c~s-his-thr-val-~l~-l~s-~l~-~l~-l~~~s II
I I( I-fI(
I2
13 1 I4
15
16
17
18
rg
zo
21 I22
-+I
k-y----HP8 HP1r
_ --+I HP15 HP16
23
1
24
251 261
I I
I I
PI’
I>
PI
In a series of potentiometric measurements, HARBURY and LOACH~J” used the pH dependence of the formal potentials for the one-electron reduction of heme peptides HP8 and HPrr to study z-axis ligand interactions. Over the pH range 4-12 several species might exist as ligands : OH-, H,O, .c-amino(lysine), imidazole (histidine), the heme ,.ring propionate group, and the a amino group of the N-terminal residue. Analogous exogenous ligands were also added. By comparison of apparent proton-linked functions with known dissociation constants for. amino acid functional groups, ligand assignments- in the various pH regions
Wilson
174
studies were carried out spectrophotometrically by and HP16. In general, histidine emerges as the most probable ligand in the physiological pH range. Evidence exists I3 for the involvement in HPII and HP15 of the a-amino groups of nterminal valine-11 in this pH range. At higher pH the c-amino groups can be involved. A comparison of the l&and binding characteristics of free imidazole and that of histidine-I8 indicates clearly that the formation of a macrocyclic ring (porphyrin plus peptide chain) makes the histidine a significantly better ligand than free imidazoleIn fact, histidine-I8 is probably a ligand at pH 4.2. Is Histidine-a6 behaves more like free were made.
Similar
\VAP,ME 1%for HP15
imidazole as expected. We have examined the electrochemistry of the above compounds at the DME and pyrolytic graphite electrodes. g Unfortunately (vi& SZ@Z) these techniques are especially vulnerable to inIIuence by aggregation effects. II-l3 All of the heme peptides are electroactive but, with the exception of HP16, give long drawn out waves. The presence of strongly coordinating ligands (histidine-I8 and -26 in HP16) or imidazole added to HPrr or HP15 tends to break up aggregation and yield polarographic waves of reasonable shape. Under these conditions all of the derivatives exhibit essentially the same electrochemical and spectral behavior_ The observed half-wave potentials were within IO mV of the potentiometric value for HP16 (see Tab. I) and no significant differences in the kinetic behavior were noted.
Table I.
Formal potentials for heme models
Compounds
1 W”
(pH 7.0)~ V
Medium
Reference
+0.256
Phosphate
I4
-0.233 -0.23 -0.19 -0.207
0.05 M Phosphate 0.05 M Phosphate -
I3 I3 =5
-0.220 -0.221 -0.210 -0.074 -0-05
b imidazole buffer b b 0.05 M Phosphate/ 0.05 M Acetate b b
! I.
2. 3_ 4. 5. 6. 7_ 5. g_
IO.
Cytochrome
c
(horse heart) HP16 HPI~ HPII HP8 Bis-his-mesoheme HP8 + imidazole Monohis-mesoheme Hismet-mesoheme HPP + n-acetyl
dZ-methionine I I. Monomet-mesoheme
12. Bis-met-mesoheme a Potentials
4.004 +0.020
given with respect
(see Experimental) b Medium -0.1
KH2P0,/K2HP0,
to the standard
hydrogen
M Phospate buffer pH 7.0 30 o/o V/V
12 16 II 16
this work I7 .
electrode
16 16 (SHE)
dioxane-water
Electrochemical
Studies
of Porphyrin
Redox
Reactions
=75
In the present work we will describe an approach t-o porphyrin model compounds for electrochemical studies in which the amino acids of interest are covalently bound to the porphyrin ring. 1*-20 The advantage of this approach over the heme peptides is that the number of possible ligands can be restricted while still maintaining a covalent linkage. Further, the approach makes possible the examination of methionine as a ligand since it is now known to be an axial &and in both oxidized and reduced cytochrome c. 1
Experimental Apparatzrs
and methods
Cyclic voltammetric data were obtained using the apparatus described previously. 21 Polarographic measurements were made at 25 OC in an H-cell constructed from a pyres cuvette which required about 1.5 cm3 of solution. Spectral measurements could also be made in this cell if desired. _4n Ag IA&l reference electrode was used for these measurementswhich had a potential of +o.zoo V ZJUS. standard hydrogen electrode (SHE). The pH measurements were made using a SARGENT miniature combination electrode standardized against NBS buffers. The apparent pH measurements in the dioxane-water mixtures were made on this basis. Materials
The synthesis of the mesoheme derivatives is described elsewhere. Is Stock solutions of these compounds were prepared in potassium phosphate buffer whose pH was adjusted with potassium hydroxide. The 30 yO (V/V) dioxane-water solutions were prepared from freshly distilled dioxane, 22 and stored in the dark. This is quite important because the peroxide impurities are both electroactive and destructive to the mesoheme derivatives .
Results and discussion Polarograplty
of mesoheme
derivatives
.
Fig. I shows the structure of one class of mesoheme derivatives Z-methionine is covalently attached to the porphyrin ring studied. through the carboxyl group of the propionic acid side chain. If the substi,tuent, R attached to the other side chain is Z-histidine, then a histidinemethionine (his-met) derivative results. rs In this way the various mesoheme compounds shown in Table I are obtained. The electrochemical experiments described below were carried out under conditions carefully defined by the rather detailed spectral studies described elsewhere. 16
y2
CH2 I
72
C”, I
Fig. I. Structure of a methionine R = histidine, methionine.
COR
CoNHiHco27”2
- mesoheme derivative. A.B = C,H,.
y2 7 C”3
The pH range (6.5-11.0) over which the measurements were made was defined by the existence of reversible one-electron waves with diffusion controlled limiting current. The pH dependence of the bis-his-derivative is shpwn in Fig. 2. The observed behavior is very similar to that obtained by HARBURY and LOACH u for HPS with added imidazole (Table I). The upward break in the curve at an apparent pH of 7 .5 is characteristic of a proton-linked function associated with the ferroheme form. In view of the fact that spectral evidence strongly suggests the esistence of a bis-histidine complex at this pH, the pH dependence reflects the protonation of the imidazole ring. Since the pK for histidine is 6.1 then there is either some steric hincirance to comples formation or alternatively the interaction of another ligand. _4t about pH II there appears to be another break in the cxve which is characteristic of an acid-base reaction involving the osidized form and probably histidine. The curve for the monohistidine case is characteristic of a high Spectral evidence suggests that the histidine is strongly spin svstem. coordinated in the oxidized and reduced forms. It should not be assumed, however, that the convergence of the mono- and bis-histidine potentials at pH 6.5 means that we are dealing with the same process. In the latter case reduction clearly occurs without change of ligands while in the former water and/or hydroxide may be involved. Preferential stabilization of the Fe(I1) o-xidation state is indicated by the dramatic shift to more positive potentials exhibited by the his-met-, mono-met- and bis-met-derivatives {see Table I>. The behavior of his-metis of particular interest because it possesses the two Iigands found in cytochrome c_ The potentiaLpI curve for the latter resembles the bis-his-
Electrochemical
Studies
of Porphyrin
Redox
Reactions
177
curve in terms of shape although it is shifted about 3.5 p_H units lower c which and over 450 IEV more positive. I4 In contrast to cytochrome exhibits low spin spectra at pH 7 as does bis-his-, the spectrum of the his-met-derivative has characteristics of both high and low spin, possibly mixed. 16 The potential of his-met at pH 7 is in reasonable agreement with that of compound IO (Table I). Above pH 7 the shape of the hismet. monomet and bismet curves reflect the decreasing ability of methionine to form a strong complex with the ferri-heme. The l&and replacing
l-E
-50
-
-100
-
-200
‘7
-250
-
I
6.0
1
I
I
I
I
7.0
8.0
9.0
10.0
11.0 PH
Fig. z. Formal (a
dependence of some mesoheme derivatives. in 30 010 (V/v) dioxane-water mixture. (A) monomet, (e) hismet, (e) monohis, (A) bishis.
potential
approximately
bismet.
2
-
X
pH
10 --1 M
Heme
concentration
Wilson
178
the methionine would be either water and/or hydroAxide. KOLSKI and PLANE ~3 have shown that the reaction H,O-FeP-H,O
+ H+ + H,O-FeP-OH
(I)
where FeP stands for the Fe(II1) derivative of deuteroporphyrin 2,4disulfonic acid [Fe(DSS)] has a pK of 7.7. The divergence of the monomet and bismet cuxres below pH 7 suggest s the increased involvement of both metbionine ligands in complex formation.
Conclllsions
It is clear from this study that the introduction of methionine as a well defined interacting ligand in place of histidine results in a positive shift in formal potential which is indicative of increased stabilization of the lower iron oxidation state. Water as a ligand also causes a positive shift which is not as great as that of met’hionine. Favorable conditions for iron methionine interactions require the formation of an Fe(U) methionine complex at a pH somewhat less than 7 where other potential ligands cannot compete effectively because they are no longer basic in nature. However, in spite of the introduction of methionine, the formal potentials for these model compounds differ greatly from cytochrome c. Such model systems apparently do not approximate the heme environment nor do they duplicate the role of the peptide chain in orienting the methionine properly. The recent elegant reconstitution studies of cyanogen bromidecleaved cytochrome c by CoRR.ADIx and HARBURY 34have suggested the possibility of further study of cytochrome c models which more fully approximate the behavior of the native protein.
Acknowledgments
We would like to thank L. P. HAGER and P. IL WARME for the generous gift of the mesoheme derivatives. This work was supported in part by National Science Foundation Grant GP-28051.
References 1
T. TXKXNO, R_ SWANSON, Harbor
2
W.&f.
O_B_ KALLAI and R.E. DICKERSON, Cold Spring Symposium on Quantitative Biology, 36, 397 (1971) CLARK, Oxidation-Reduction Polenfiak of Organic Systems, Williams
and WiMns,
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