Coupling between Oxidation State and Hydrogen

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 10, Issue of May 25, pp. 5052-5057, 1981 Printed in U S. A.

Coupling between Oxidation State and Hydrogen Bond Conformation in High Potential Iron-Sulfur Protein* (Received for publication, May 15, 1980, and in revised form, September 30, 1980)

Robert P. Sheridan:, Leland C. Allen§, and Charles W. Carter, Jr.¶11 From the Department of Chemistry, Princeton University, Princeton, New Jersey 08544 and 1 Department of Biochemistry and Nutrition, University of North Carolina, Chapel Hill, North Carolina 27514

* This work was supported by Grant GM 26462 (L. C. A.) from the National Institutes of Health and Grant PCM-08138 (C. W. C.) from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. t Present address, Institute for Cancer Research, Philadelphia, PA

19111. § To whom reprint requests should be addressed. 1 Jefferson-Pilot Fellow in Academic Medicine for the duration of this work.

Iron-sulfur proteins transport electrons in numerous biological oxidation-reduction processes, including photosynthetic and oxidative phosphorylation and nitrogen fixation. Iron is always tetrahedrally coordinated by sulfur in these proteins, the number of irons per molecule being 1, 2, 3, 4, or 8 depending on the class. Proteins (all X2 \

H

1 Xi

02 X2

83 L

i

X

O02

N-S DISTANCE C-N-S ANGLE O-C-N-S DIHEDRAL ANGLE N-S-H2 ANGLE C-N-S-H2 DIHEDRAL ANGLE N-S-X2 ANGLE +1/-1 ABOVE/BELOW PLANE OF N,S,X1 +1/ 1 ABOVE/BELOW PLANE OF S,XI,X2

FIG. 3. Formamide-SX3 dimer with internal coordinates defined. This is a model for NH...S* hydrogen bonds in iron-sulfur proteins. Formamide represents the peptide backbone, S = S*, and Xi, X2, X3, replace Fe. H H

N-

O-C 01

TABLE I Internal coordinates for the hydrogen bonds in HiPIP 01 r (A) 02 Xl Hydrogen bond x2 (deg3 (deg)

H

1 Xi

02

X2

N-OH DISTANCE C -N-OH ANGLE O-C-N-OH DIHEDRAL ANGLE

N-OH-CZ

a

H

Y

ANGLE

C-N-OH--CZ DIHEDRAL ANGLE

X3 N-OH-CZ-CEI DIHEDRAL ANGLE FIG. 4. Formamide-phenol dimer with internal coordinates defined. This is a model for the NH 72 0i Tyr 19 hydrogen bond in HiPIP. The nomenclature for the atoms in the phenol ring is that for tyrosine used by the Brookhaven Protein Data Bank: OH = 0, CD1 = ClI, HH = Hn. A variable point charge at Y is placed near the ring to model the influence of S*3.

dimers. In the model dimers H2 replaces C,; Xi, X2, and XA3 replace Fe atoms.

Monomer Geometries-Monomer geometry for formamide is the same as employed previously (14) and is kept fixed unless otherwise stated. The geometry of the SRX monomers were taken from the xray structures of various model compounds (17-19). For the SHX monomer (R = -H), the S-H distance is taken from SH2. The atom X is set at an H-S-R angle of 1070 (the average Cp-S,-Fe angle for oxidized HiPIP) and at a distance of 1.33 A, the minimum energy distance for Zx = 1.0 (calculations are not sensitive to changes in this

NH 48- . . Sy 46 Oxidized Reduced NH 81 .* *Sy 46 Oxidized Reduced NH 65- Sy 63 Oxidized Reduced NH 79 .. S-y 77 Oxidized Reduced NH 77-..S*1 Oxidized Reduced NH 49 ... S*2 Oxidized Reduced

3.676 3.539

122 127

-143 -172

106 104

33 49

113 106

-1 -1

3.684 3.599

113 116

165 -178

106 105

112 98

123 123

1 1

3.522 3.383

124 121

-160 -166

100 105

56 56

145 146

-1

3.472 3.406

141 138

-171 179

102 105

1 7

120 115

-1 -1

3.661 3.476

106 116

132 159

107 102

-38 -56

152

150

1 1

-1 -1

4.005 3.904

98 92

84 83

171 170

120 131

100 100

1 1

1 1

X2

(deg)

r

NH 72.. 0-0 19 Oxidized Reduced

j

(A)

3.017 3.096

01

XI

02

123 123

175 -177

140 143

59 50

33 35

-1

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0 C

distance). For the SX3 monomer, the X1-S-X2 = XI-S-X3 = X2-S-X3 angles are set to 750 (the average Fe-S*-Fe angle in HiPIP) and at the minimum energy distance of 1.37 A (for Z, = 1.0). The phenol geometry is taken from small molecule crystallography (20) and the hydroxyl hydrogen H, is assumed to be in the plane of the ring. A point charge (position Y in Fig. 4) is placed 2.63 A from CD1, at an angle of 950 to CE1, and a dihedral angle of 1370 to CZ. All of these values are derived from the position of S*3 relative to the Tyr 19 ring. Dimer Geometries-The relative orientations of formamide and SHX, SX3, or phenol in the hydrogen bonded dimers are fixed using the internal coordinates derived from the x-ray coordinates as listed in Table I. The N-Hi distance, rl, is unavailable from crystallography and is obtained by energy minimization. Effect of Change in Cluster Oxidation State-The effect of a change in oxidation state on the hydrogen bond energy is modeled by varying the nuclear charge, Zx, while keeping the geometry fixed. For Zx ranges between 0.0 and 1.0, for SX3 between 0.333 and 0.667. For phenol the point charge has a value of 0.0 to -1.0. Since the number of electrons remains constant, the values of Zx = 0.0 and 0.333 correspond to the fully anionic dimers and the values Zx = 1.0 andu 0.667 correspond to the neutral dimers. It is expected that the actual charge on the atoms involved in the hydrogen bonds in the proteins will fall between these limiting cases, the reduced forms being closer to the anionic and the oxidized forms being closer to the neutral. Similarly for formamide-phenol, a point charge of -1.0 corresponds to the polarized ring and a 0.0 point charge to the unpolarized ring, representing Tyr 19 in the reduced and oxidized states, respectively. Calculations-Ab initio calculations were made by the SCF-MOLCAO method using the STO-3G basis set with a modified version of Gaussian 70 (21). A hydrogen ls basis orbital was assigned to X. For each substituted monomer as well as for the hydrogen bonded dimers, we calculated the energy for the neutral and anionic limiting cases. The dimers were computed at their optimized (minimum energy) and experimental geometries. We have recently discussed the detailed electronic nature of this type of hydrogen bond (22). The difference in total energy between neutral and anionic forms of the substituted monomers: A ET = ET(anion) - ET(neutral) gives their relative stabilities. The lower the value of A ET, the more stable the anionic form relative to the neutral form. In other words, lower AET'S imply more effective delocalization of charge. Dimerization energies (ED'S) were calculated by subtracting the energies of SHX, SX3, or phenol and optimized formamide monomers from the energy of the dimer. More positive values of ED indicate stronger hydrogen bonds. Calculations with intermediate values of Zx (for a fixed geometry) indicate that ED varies nearly linearly with Zx.

Hydrogen Bonds in HiPIP TABLE II Dimerization energies (kcal/mol) N(ut=10) (Zx = 0.) Hydrogen bond geometry

NH 48 ... Sy 46 Oxidized Reduced Reduced - oxidized NH 81 ... Sy 46 Oxidized Reduced Reduced - oxidized NH 65 ... Sy 63 Oxidized Reduced Reduced - oxidized NH 79... Sy 77 Oxidized Reduced Reduced - oxidized

X-S-R R= neutral

11.4 20.2 8.8

10.0 17.4 7.4

1.9 2.6 0.7

15.6 19.2 3.6

13.7 16.6 2.9

AET

1.9 2.0 0.1

18.0 22.7 4.7

16.1 20.7 4.6

10 KCAL/MOL

1.7 2.2 0.5

18.3 21.5 3.2

16.6 19.3 2.7

(Zx 0.667) (Zx

=

8.6 17.9 9.3

7.1 16.3 9.2

0.2 0.1 -0.1

1.3 0.8 -0.5

1.1 0.7 -0.4

6.8 6.0 -0.8

2.2 2.3 0.1

RESULTS AND DISCUSSION

Electron-withdrawing Substituents and NH...S HydroBonds Can Stabilize Reduced Cluster Oxidation

gen

States-The difference in total energy between neutral and anionic forms, AET, for model complexes HN X-S \

R

-H +OPTIMIZED

H

is rather strongly influenced by the nature and environment of the sulfur substituent (Fig. 5). The AET ordering for substituents, aliphatic > -CH2-C6H5 > -C6H5 > -C6H4NO2, is parallel to the order of midpoint potentials measured experimentally for model compounds using the same substituents (Table II, Ref. 15). Small AET values correspond to high midpoint potentials. Experimentally it is found that model compounds with more electron-withdrawing substituents have higher potentials.3 The ability to mimic this by the trend in

'The absolute spacing between values of A ET for various substituents in Fig. 5 is dependent on our choice of anionic and neutral limiting cases and also on the basis set used in our calculations. For a given pair of limiting cases AET for a given substituent appears insensitive to rotation around single bonds (variance -CH3 > case, AET = ET(-1.0) -CH2-CH3 > -C(CH3)3. For AET = ET-0.2) ET(neutral) the order is -C(CH3)3 > -CH2-CH3 > -CH3 > -H, consistent with the -

-

H-BOND.

.0

-

N

'b~~

FIG. 5. The relative abilities of various sulfur substituents and hydrogen bonding to stabilize a negative charge. The vertical scale AET represents the difference in total energy between anionic and neutral SRX monomers for different R groups. Lower AET's represent more effective delocalization of negative charge. The aliphatic substituents (-H, -CH3,-CH2-CH3,-C(CH3)3) are treated as a group because the ordering within the group depends on the choice of limiting cases. See footnote 3.

AET values supports the comparisons given below. Stabilization afforded by an optimized NH... S hydrogen bond to sulfur is comparable to that of the most electronwithdrawing substituents studied (R = -C6H5, -C6H4NO2). This result reinforces frequent suggestions (1-4, 11-13) that protein backbone N-H groups within hydrogen bonding distance of cluster sulfur atoms raise the intrinsic reduction potential of the (RS)4Fe4S4* cluster by electrostatic stabilization of added negative charge in reduced, relative to oxidized forms of the cluster. Moreover, it favors a previous interpretation that the increased reduction potential observed for R = (acetyl cysteine NH methyl) in nonaqueous media (Ref. 16, Table II) does arise chiefly from the NH. .S* hydrogen bonds from ligand amide nitrogens (4). It should be noted that AET for the computed minimum increasing rmidpoint potentials of model compounds. The incorrect ordering when we use limiting cases with very different net charges occurs because the electron donating property of methyl groups is swamped by the delocalization of the extra charge, which as usual, leads to lower energy for larger substituents. Within the group of aliphatic substituents the order of midpoint potential is -C(CH3)2 > -CH(CH3)2 >-CH2-CH3 >-CH3 because the potential is governed by electron donation from methyl groups; the larger the number of methyls the less the ability of the substituent to delocalize charge. Our study of the ordering at different values of charge, combined with our comparision of the differences in AET with the differences in midpoint potential, suggest that the actual amount of extra charge delocalized in the model compounds is rather small, c 0.4 rather than 1.0. Fortunately, the trend in AET aliphatic > -CH2-C6H5 > H-bond > -C6H5NO2 (due to the strong effect of resonance delocalization) is independent of limiting case. For a given substituent on the sulfur electron donor, relative ordering of H-bond strength is also independent of limiting case (for the H-bond calculations R = -H is used as the substituent on sulfur).


1.5 1.6 0.1

4.6 3.7 -0.9

and

T

Anion 0.333) neutral

Unpolarized Polarized Polarized (Y = 0.0) (Y = -1.0) unpolarized

X-S

D

Anion

=

NH 72. ... 0i 19 Oxidized Reduced Reduced - oxidized

I

1.4 2.8 1.4

Neutral

NH 77.. S* 1 Oxidized Reduced Reduced - oxidized NH 49 .. S*2 Oxidized Reduced Reduced - oxidized

5055

5056

506Hydrogen Bonds in HiPIP


formamide-SHX reference hydrogen bond shown in Fig. 5 to the HiPIP tertiary structure through the intermediacy of represents the theoretical maximum stabilization, i.e. a hydro- these hydrogen bonds. That is, more highly negative charges gen bond fixed at an experimental geometry would show differentially stabilize the more favorable geometries. slightly less stabilization. Of all the systems calculated here, Coupling of Oxidation State with Geometry of the NH O 19 Hydrogen Bond-Two separate but mutually the SHX reference monomer, like the other aliphatic mono- 72. ..O mers, allows little delocalization of charge on the substituent. reinforcing effects couple the ring of Tyr 19 to the cluster Therefore, it will act as a good electron donor, and hydrogen oxidation state. The first is the purely electrostatic interaction bonds formed with it will be strong. Extra stabilization due to of the ring with the cluster (independent of the hydrogen hydrogen bonding for the other substituents (i.e. R = -C6H5, bond), and the second is the polarization of the ring by the -C6H4NO2) studied here will be less pronounced because as cluster which makes the ON a better electron donor to the NH the charge on sulfur is more effectively delocalized by substit- 72... O** hydrogen bond. uents, less charge is available to be further delocalized in a Calculations on the perturbation of SX,3 (our model for S*) hydrogen bond. In particular, water-to-sulfur hydrogen bond- on the electronic distribution of phenol in the absence of the ing should show a more dramatic effect on the reduction hydrogen bond show that the interaction is purely electropotentials of analog clusters where R = aliphatic than on static and the use of a point charge at Y to replace SX3 is those for which R = aromatic (or AcCysNHMe). Such a trend justified. The electrostatic potential around phenol is qualiis apparent (15, 16), although data for several key examples tatively such that a negative charge near CS, at 450 from the involving aromatic substituents were not obtained for techni- ring feels a positive field from phenol and hence is attracted cal reasons. to the ring. The energy of interaction of the point charge with In summary, the calculations represented in Fig. 5 indicate phenol is 3 keal/mol for a -1.0 point charge and 0 keal/mol that both the nature of sulfur substituents and the proximity when the point charge is 0.0. The fact that the point charge of hydrogen bonding groups can significantly affect the induces a dipole in the ring enhances the attractive energy stability of charge on the sulfurs. We would expect pro- only slightly over that of an unpolarized phenol. tein cysteinate sulfurs to behave similarly to aliphatic-substiWe now consider the effect of polarization on the hydrogen tuted sulfurs and thus to be rather good electron donors in bond. Dimerization energies for the formamide-phenol dimer NH. . -S hydrogen bonds. show that the polarization of phenol by a point charge at the Coupling of Oxidation State and Hydrogen Bond Geome- location of S*3 would increase the electron donating power of try-We now assess whether or not the hydrogen bond ge- the hydroxyl oxygen, On, for any fixed geometry.4 Thus, the ometries actually observed in HiPIP are sufficiently favorable ED increases by approximately 2 keal/mol with a change in to constitute functionally significant interactions. The amount point charge magnitude from 0 to -1. (It should be noted that by which a NH... S hydrogen bond might stabilize the re- ED values for the NH 72...O,, 19 hydrogen bond cannot be duced oxidation state relative to the oxidized (i.e. the amount directly compared with those for the NH ... S hydrogen bonds by which it can raise the oxidation-reduction potential) should because of the difference in limiting case models.) As noted (11), either of the attractive forces on Tyr 19 could be determined by the difference between the strength of the hydrogen bond in the two oxidation states. Table II shows the account for movement of the ring closer toward the cluster in results of calculating ED, the dimerization energy, for form- reduced HiPIP. Our quantitative estimates for the strength of amide-SHX and formamide -SX3 models configured with ge- these interactions strengthens the proposal (4) that a charge ometries of NH ... S hydrogen bonds observed in reduced and induced-dipole interaction, supported by the NH 72-0. O, 19 oxidized HiPIP. hydrogen bond, exerts a reciprocal perturbation on the (CysCalculations of ED were carried out for each geometry with S)4Fe4S4* cluster. Zx fixed first at 0.0 and then at 1.0 for the SHX case or 0.333 Note Added in Proof-Sweeney and Magliozzo (23) have recently and 0.667 for SX3. The relative strengths of hydrogen bonds shown that deuteration of the NH ... S H-bonds in ferredoxin from in HiPIP, estimated by ED values in Table II, vary signifi- C. pasteurianum causes a small lowering of the oxidation-reduction cantly with changes in geometry and substantially with potential. They argue that deuteration should strengthen the Hchanges in nuclear charge (Zx). ED values are 7 to 20 keal/ bonds and raise the potential, contrary to the observations; they mol greater in anionic than in neutral dimers, and the mag- suggest, therefore, that H-bonds are not important modifiers of the in iron-sulfur proteins. They point out, however, that if all nitude of the change in EDcaused by a change in Zx is greater potential the potential wells for the NH ... S bonds were broader in the reduced in every case but one (NH 49... S*2) for the geometry ob- than in the oxidized protein, the expected deuteration effect would be served in reduced HiPIP. Changes in hydrogen bond geometry cancelled or reversed and their results would then not rule out the observed in the crystal structures of reduced and oxidized influence of H-bonds. In our calculations with fixed experimental HiPIP cause changes in ED of as much as 1.4 keal/mol when geometries hydrogen bond strength is a function of N-H distance. Zx = 1.0 (0.667) and 9.3 keal/mol when Zx = 0.0 (0.333). This potential well of ED versus proton position is much broader in anionic dimers (our model for H-bonds in the reduced protein) Variation in hydrogen bond strength with geometry as well as the than in neutral dimers (our model for H-bond in oxidized protein). with charge is consistent with the general properties of hydro- Thus our calculations reveal a special situation analogous to the one gen bonds as discussed previously (14). This variation is large discussed by Sweeney and Magliozzo. It is not unreasonable that the enough to couple hydrogen bond geometry with oxidation situation could hold for the hydrogen bonds in iron-sulfur proteins, state. It is worth noting in this regard thataNH. *S . hydrogen and thus the results of these authors do not rule out the influence of * S H-bonds on the oxidation-reduction potential. bond with especially poor geometries (e.g. NH 49... S*2) NH. shows very small variation with charge; such hydrogen bonds For reasons having to do with imposition of regularized geometry would contribute very little to raising the potential. on the reduced HiPIP atomic coordinates, changes in the N ...O, and The dimerization energy differences ((anion - neutral) and C1 ...S*3 distances between oxidized and reduced HiPIP are less (reduced - oxidized), TableII) show in each case (excepting than previously reported on the basis of refined but unregularized NH 49... S*2) a significant coupling between the geometry reduced HiPIP coordinates, wherein the N ...O0N and C1 ... 5*3 distances shorted by about 0.3 A and 0.2 A (11). Our calculations, and charge effects; the change in H-bond energy for a given therefore, show significant difference between the change in charge is considerably greater for the reduced two geometries nofor energetically the same charge at Y, our model for S*3 (see geometry than for the oxidized geometry. This observation is TableII). Magnitudes of the actual geometry changes, if any, remain precisely that expected if the cluster oxidation state is coupled to be established by further crystallographic studies.

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