A Synthetic Analogue for the Active Site of Plant-Type Ferredoxin: Two Different Coordination Isomers by a Four-Cys-Containing [ 201 -Peptide '

'

N O R I K A Z U UEYAMA, SATORU UENO,' AKIRA NAKAMURA, * KElSHlRO WADA,3 HIROSHI MATSUBARA,3 SHIN-ICHIRO KUMAGA1,4 SHUMPEI SAKAKIBARA,4 and TOMITAKE TSUKIHARA5

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Departments of Macromolecular Science and 'Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560; Departments of *Pharmaceutical Sciences and 5Chemical Engineering, Tokushirna University, Tokushima 770; and 4 P r ~ t e i nResearch Foundation, Ina, Minoh-shi, Osaka 562, Japan

SYNOPSIS

The ( FezSz) '+ complex of an artificial 20-peptide ligand, Ac-Pro-Tyr-Ser-Cys-Arg-AlaGly-Ala-Cys-Ser-Thr-Cys-Ala-Gly-Pro-Leu-Leu-Thr-Cys-Val-NHz, containing an invariant Cys-A-B-C-D-Cys-X-Y-Cys(A, B, C, D, X, Y = amino acid residues) fragment of planttype ferredoxins was synthesized by a ligand exchange method with [ Fez&( S-t-Bu),] '-. 'H-nmr spectroscopic and electrochemical data of the complex indicate the presence of two coordination isomers. One of them having a Cys-X-Y-Cysbridging coordination to the two Fe(II1) ions, has the (FezSZ)*'core environment similar to those of the denatured plant-type ferredoxins and exhibits a positive shifted redox potential at -0.64 V vs saturated colonel electrode (SCE) in N,N-dimethylformamide (DMF). Another isomer with the Cys-A-B-C-D-Cysbridging coordination shows a negative redox potential at -0.96 V vs SCE in DMF. 0 1992 John Wiley & Sons, Inc.

INTRODUCTION Plant-type ferredoxins, which occur widely in algae and higher plants, have approximately 100 amino acid residues and 1 (Fe2S2)2' core. These ferredoxins play important roles in photosynthesis, in particular, for reduction of NADP' in cooperation with ferredoxin-NADP' oxidoreductase.' Native spinach ferredoxin has a redox potential at -0.42 V (vs. N H E ) ,which corresponds to a n approximate value, -0.66 V [vs saturated calomel electrode ( S C E ) ] .2-4 This value is important for one of the components existing in the electron transfer chains in photosystem I. Many synthetic model complexes of plant-type '-(S,-o-xyl = oferredoxins, e.g., [Fez&( S2-o-xyl)2] xylene-a,@'-dithiolate) , have been synthesized by Holm's g r o ~ p .These ~ , ~ complexes exhibit two characteristic absorption maxima presumably due t o a ligand-metal charge transfer absorption, for ex-

Biopolymers, Vol. 32, 1535-1544 (1992) 0 1992 John Wiley & Sons, Inc.

CCC 0006-3525/92/111535-10

ample, at 414 and 453 nm for [ Fe2S2( S2-o-xy1)2]2in N,N-dimethylformamide (DMF) . These maxima are different from those observed for the native proteins a t 423 and 466 nm. Simple peptide model complexes also have two absorption maxima a t 423 and 453 nm ( s h ) for [ Fe2S2{ Ac-Gly2-( cys-Gly2)2NH2)2]2-,7in Me2S0 or a t 417 and 450 nm for 2- ( Z = benzyl[Fez& (Z-~ys-Ala-Ala-cys-OMe)~] oxycarbonyl) in DMF.' These values do not coincide with those found for the native proteins. The CD spectra of [ Fe2S2( Z-cys-Ala-Ala-cys-OMe ) 2 ] 2- and [Fe2S2(Z-Ala-cys-OMe)4]2- were found to be different from that of the native Spirulina maxima ferred~xin.~ T h e simple synthetic model complexes generally exhibit extremely negative redox potentials, for example, -1.50 V (vs SCE) for [Fe2S2(S2-o-xy1)2]2-. Previously we reported the positive shift of redox potential (-1.06 V vs S C E ) of [Fe2S2(Z-cys-AlaA l a - ~ y s - O M e ) ~ ]in DMF that has two bidentate chelating peptide ligands coordinating to Fe( 111) However, there is still a large difference in redox potential between the tetrapeptide complex and the native plant-type ferredoxin.

'-

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UEYAMA ET AL.

Such spectral and electrochemical differences between the synthetic analogues and the native planttype ferredoxins are caused by the unique environment of a peptide chain surrounding an (Fe,S,) *+ core. The spectral and electrochemical properties thus depend on the amino acid residues in the vicinity of the (Fe,S,)" core. Therefore, a peptide with a sequence of amino acid residues which is invariant in [ 2Fe-2SI ferredoxin is important in this respect. Actually, four Cys residues, ligating (Fe,S,) core in Spirulina platensis ferredoxin, are located in a small domain separated from a major domain, as illustrated in Figure 1.A 20-peptide containing four Cys residues as illustrated in Figure 2 was thus designed in consideration of the preferable conformation." In this paper, we present the synthesis and the spectral and electrochemical properties of a 20-peptide/ (Fez&)*+ complex designed on the basis of the conformational energy calculation and the x-ray structure analysis of S. platensis ferredoxin."

Ac-Pro -1vr - S e r - 0 s - A r g -Ala -Gly -Ala -Cys - S e r

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T h r - C y s - A l a - G I y - P r o - L e u - L e u - T h r - C y s - V a l -NH2 20-pep

'+

EXPERIMENTAL Materials Boc-Ala-OH (Boc = t-butoxycarbonyl) , Boc-Arg(Tos) -OH (Tos: tosyl) , Boc-Leu-OH H20, Boc-ProOH, Boc-Ser (Bzl)-OH (Bzl: benzyl) , Boc-Thr ( Bzl) OH, Boc-Leu-ONSu (NSu: succimido) , Boc-ProONSu, Boc-Ser (Bzl) -ONSu, Boc-Tyr [Bzl (Cl),] ONSu (Bzl(Cl)z:2,6-dichlorobenzyl), Boc-Thr (Bzl)ONSu, Boc-Ah-Gly-OBzl, trifluoroacetic acid (TFA) , anhydrous hydrogen chloride in dioxane (4.5 and 4.8 N ) , N,N'-dicyclohexylcarbodiimide (DCC) , N-hy-

Figure 2. Artificial 20-pep ligand prepared by the solution method and a designed structure of the ( Fez&)*+/ 20-pep complex by the energy-minimized calculations."

droxybenzotriazole ( HOBt ) , acetic acid, N-hydroxysuccinimide ester ( AcONSu) , and 1-ethyl-3-(3-diethylaminopropyl) carbodiimide ( WSCI) were obtained from Protein Research Foundation, Osaka. All solvents were purified by distillation before use. All the other reagents were of commercial grade. Native spinach ferredoxin was prepared by the same procedure reported in the literature.'

Peptide Synthesis The peptide synthesis were performed through the following coupling among Ac-I, 11, and I11 fragments by the liquid phase condensation method.

Figure 1. The polypeptide chain of S. platensis ferredoxin. The circle part shows the active site of the ferredoxin."

Boc-Ala-Gly-OPac(Pac = Phenacyl). T o a solution of Boc-Ala-Gly-OBzl (50.4 g, 0.15 mol) in MeOH (150 m l ) was added 90 mL of 2 N NaOH aq. (0.18 mol). After stirring for 30 min, the solution was concentrated under reduced pressure and the residue was dissolved in water. T h e aqueous solution was

SYNTHETIC ANALOGUE FOR PLANT-TYPE FERREDOXIN

washed twice with AcOEt. After addition of 1N HCI (180 mL, 0.18 mol) and NaC1, the product was extracted with AcOEt, dried by benzene azeotrope, and concentrated under reduced pressure. T h e residue was recrystallized from THF/AcOEt/hexane to give Boc-Ala-Gly-OH. Boc-Ala-Gly-OH ( 26.1 g, 106 mmol) and phenacyl bromide (22.5 g, 140 mmol) were dissolved in DMF (300 m L ) . Triethylamine (16 mL, 140 mmol) was slowly added to the stirred solution and the solution was concentrated in vacuo. T h e residue was dissolved in AcOEt. The extract was washed with HzO, 1 N HCl aq., HzO, 5% NaHC03 aq., and H20,dried over Na2S0,, and concentrated. The crude product was recrystallized from AcOEt/ether to give Boc-Ala-Gly-OPac (yield 27.0 g, 70%); mp 108-109°C. [ a ]-30.6" ~ (c, 0.633 in MeOH). Anal. Calcd. for C18H24N206: C, 59.33; H, 6.63; N, 7.69. Found: C, 59.31; H, 6.63; N, 7.68. Boc-Arg (10s)-Ah-G/y-OPac (1-3). Boc-Ala-GlyOPac (3.7 g, 10 mmol) was dissolved in TFA (20 ml) a t -6°C. The reaction mixture was concentrated to about a half volume under reduced pressure. Then 4.5 N HCl/dioxane ( 3 mL, 14 mmol) was added to the solution. All solvents were evaporated in vacuo. The residue was washed with ether, dried over NaOH pellets in vacuo. T o a solution of the deblocked peptide in DMF ( 150 m L ) was added HOBt ( 1.42 g, 10.5 mmol) and Boc-Arg( Tos) -OH (4.5 g, 10.5 mmol). WSCI ( 2 mL, 10.5 mmol) was added to the solution with stirring a t -20°C. After the reaction, the solution was concentrated under reduced pressure. T h e residue was dissolved in CHC13. The extract was washed with HzO, 1 N HC1 aq., H 2 0 , 5% NaHC03 aq., and H20,dried over Na2SO4,and concentrated and followed by two precipitations from AcOEt/ether to give the titled compound (yield 4.9 g, 73% ) . [ a ] n-21.4" (c, 0.8278 in MeOH) . Anal. Calcd. for C31H42N609S0.8H20: C, 54.03; H, 6.38; N, 12.19. F o u n d C, 53.99; H, 6.35; N, 12.26.

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Boc-Cys (Aem) -Arg (Tos)-Ah-Gly- OPac (1-4). Deblocked fragment 1-3 was dissolved in DMF (150 m L ) and HOBt (0.94 g, 7 mmol), and BocCys ( Acm) -OH (Acm = acetamidomethyl) ( 2.02 g, 7 mmol) was added t o the solution. WSCI ( 1.27 mL, 7 mmol) was added t o the stirring solution a t -20°C and stirred for 3 h at room temperature. The isolation of fragment 1-4 was carried out by the same method for the synthesis of fragment 1-3and purified twice from CHCl,/AcOEt to give white powder (yield 3.5 g, 63%). [ a ]-26.6" ~ (c, 1.047 in MeOH) . Anal. Calcd. for C37H52N8-011S20.7H20: C, 51.58; H, 6.25; N, 13.01. F o u n d C, 51.55; H, 6.21; N, 12.91.

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Boc-Ser (Bz1)-Cys (Acm) -Arg (Tos)-Ala-Gly-OPac (15 ) . Fragment 1-5 was synthesized by the condensation of deblocked fragment 1-4 (2.8 g, 3.3 mmol) and Boc-Ser( Bzl) -0NSu (1.3 g, 3.3 mmol) by the same procedure for the synthesis of fragment 1-3, and purified with the precipitation from MeOH/diethyl ether (yield 3.27 g, 97%) . [ aID-10.6" (c, 0.462 in D M F ) . Anal. Calcd. for C47H63N9013S2 0.8H20: C, 55.14; H, 6.19; N, 12.28. F o u n d C, 54.26; H, 6.19; N, 12.18.

Boc- Tyr [ B z (C1)2] - S e r ( B z l ) - Cys (Acm)- Arg (Tos)Ala-Gly-OPac (1-6). Fragment 1-6 was synthesized by the condensation of deblocked fragment 1-5 (3.2 g, 3.1 mmol) and Boc-Tyr [ Bz( Cl),] - 0 N S u (1.67 g, 3.1 mmol) by the same procedure described above (yield3.6g,87.1%). [.i]o-8.00 (c, 1 . 0 6 1 i n D M F ) . Anal. Calcd. for C63H76N10015S2C12: C, 56.12; H, 5.68; N, 10.39. Found: C, 54.08; H, 5.81; N, 10.95. Boc- Pro- Tyr[Bz(CI)Z]- Ser(Bz1)- Cys(Acm)-A rg(1os)Ala-Gly-OPac (1-7). Fragment 1-7 was synthesized by the condensation of deblocked fragment 1-6 ( 3 g, 2.2 mmol) a n d Boc-Pro-ONSu (0.69 g, 2.2 mmol) by the same procedure described above. The product was precipitated from CHC13/ether and purified by silica gel chromatography ( CHC13/ MeOH / AcOEt 85:15:5) (yield 2.86 g, 90% ) . Ac- Pro - Tyr[Bz(C/2)] - Ser(Bz/) - Cys(Acm)Arg(Tos)Ah-Gly-OPac (Ac-1-7). Fragment 1-7 (700 mg, 0.48 mmol) was carried out by the same method as described for the synthesis of Ac dissolved in TFA ( 3 ml) a t -6°C. After stirring for 20 min with cooling, the reaction mixture then stirred a t room temperature. The reaction mixture was concentrated under reduced pressure. The residue was washed with ether, dried over NaOH pellets in vacuo. The residue, Et3N (0.067 mL, 0.48 mmol) and Ac-ONSu (82.9 mg, 0.53 mmol) were dissolved in 20 mL of DMF. After the reaction, the solution was concentrated under reduced pressure. The residue was washed in 1 / 3 N HC1 a n d HzO and dried over Pz05in vacuo to give the title compound (yield 630 mg, 95% ) . [ a ] ~ -25" (c, 0.359 in D M F ) . Anal. Calcd. for Cs5H77N11015S2C12 2.3H20: C, 54.64; H, 5.76; N, 10.78. F o u n d C, 54.67; H, 5.58; N, 10.70. Boc-Cys(Acm)-Ala-Cly-OPac (11-3). Boc-Ala-GlyOPac (13.5 g, 37 mmol) was dissolved in TFA (60 m L ) a t -6°C. The reaction mixture was concentrated to about a half volume under reduced pressure, 4.8 N HCl/dioxane (12 mL, 56 mmol) was added to the solution. All solvents were evaporated

1538

UEYAMA ET AL.

in vacuo. T h e residue was washed with ether, dried over NaOH pellets in vacuo. T h e residue was dissolved in DMF (300 ml) together with HOBt (5.5 g, 40.7 mmol) and Boc-Cys (Acm) -OH ( 11.8 g, 40.7 mmol). WSCI (7.5 ml, 40.7 mmol) was added to the stirring solution a t -20°C and after the reaction, the solution was concentrated under reduced pressure. The residue was dissolved in AcOEt. T h e extract was washed with H20, 1 N HC1 aq., H 2 0 , 5 % NaHC03 aq., and HzO, dried over anhydrous sodium sulfate, and the solution was concentrated under reduced pressure and followed by two precipitations from MeOH/diethyl ether t o give the titled compound (yield 17.5 g, 78%). [ a]D -30.0" (c, 0.983 in D M F ) . Anal. Calcd. for C24H34N408S 0.15H20: C, 53.25; H, 6.39; N, 10.35. F o u n d C, 53.25; H, 6.49; N, 10.32.

and after the reaction, the solution was concentrated. T h e residue was dissolved in AcOEt. T h e extract was washed with H 2 0 , 1 N HC1 aq., H20, 5% NaHC03 aq., and HzO, dried over anhyrous SOdium sulfate: concentrated and recrystallization from AcOEt/ether to give the titled compound (yield 16.0 g, 50% ) . [ a]D -2.0" (c, 0.928 in MeOH) Anal. Calcd. for C10H20N203:C, 55.54; H, 9.27; N, 12.95. Found: C, 55.62; H, 9.27; N, 12.95.

Boc-Thr (Bz1)-Cys(Acm)-Ala-G1y-OPac (I I 4 ) . Fragment 11-4 was synthesized by the reaction from deblocked fragment 11-3 (16 g, 30 mmol) and Boc-Thr(Bz1)-OH (9.7 g, 31.5 mmol) using the same procedure for 11-3 (yield 15.8 g, 72% ). [ cY]D -29.3" (c, 0.996 in MeOH). Anal. Calcd. for C35H47N5010S. 0.5H20: C, 56.90; H, 6.55; N, 9.48. Found: C, 56.89; H, 6.49; N, 9.41.

Boc- Thr (Bz1)-Cys (Arm) -Va/-NH, (111-3). Fragment 111-3 was synthesized by the same method for 111-2 (yield 9.3 g, 74% ) . [ a ] D -15.6' (c, 1.182 in MeOH) . Anal. Calcd. for C27H43N507S.2Hz0: C, 52.49; H, 7.67; N, 11.34. F o u n d C, 52.25; H, 7.61; N, 11.14.

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Boc-Ser (Bz1)-Thr (Bz1)-Cys(Acm)-Ala-Gly-OPac (II5). Fragment 11-5 was synthesized by the same pro-

cedure as described for fragment 11-3 (yield 12.7 g, 70%). [a]D -6.0" (c, 0.745 in D M F ) . Anal. Calcd. for C4&&6012S. 0.5H20: C, 59.00; H, 6.49; N, 9.17. Found: C, 59.00; H, 6.42; N, 9.08.

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Boc-Cys (Acm)-Ser (Bzl) Thr (Bz1)-Cys (Acm)-AlaGly-OPac (11-6). Fragment 11-6 was synthesized by the same procedure as described for fragment 11-3 (yield 11.2 g, 80% ). [ c y ] ~-0.3" (c, 0.819 in D M F ) . Anal. Calcd. for C51H68N8014S2 * 0.6H20: 56.09; H, 6.39; N, 10.26. F o u n d C, 56.13; H, 6.29; N, 10.07.

c,

Boc-Ala-Cys (Acrn) -Ser (621)- Thr (Bzl) -Cys (Acm) Ala-Gly-OPac (11-7). Fragment 11-7 was synthesized by the same procedure for fragment 11-3 (yield 7.96 g, 7 2 % ) . [ a ] D -6.1" (c, 1.60 in D M F ) . Anal. Calcd. for C54H73N9015S2 * H20: C, 55.42; H, 6.46; N, 10.77. Found: C, 55.37; H, 6.51; N, 10.87. Boc-Val-NH, (111-7). Boc-Val-OH (32.7 g, 150 mmol), HOBt ( 6 g, 44 mmol), and NH4HCOB(13 g, 164 mmol) was dissolved in CH2C12/DMF (400 mL, 5 : 1 v / v ) . DCC (34.0 g, 164 mmol) in CH2C12 (30 m L ) was slowly added to the stirring solution

Boc-Cys(Acm)-Val-NH, (111-2). Fragment 111-2 was synthesized from deblocked 111-1( 10.8 g, 50 mmol) and Boc-Cys ( Acm) -OH ( 15.3 g, 52.5 mmol) by the same procedure as described for the synthesis of II3 (yield 8.8 g, 45%). [a]D -27.0" (c, 0.875 in MeOH). Anal. Calcd. for C16H30N405S: C, 49.21; H, 7.74; N, 14.35. Found: C, 45.37; H, 7.29; N, 13.23.

Boc-Leu-Thr(Bzl)-Cys(Acm)-Val-NH2(III4 ) . Fragment 111-4 also synthesized by the same procedure as described for the synthesis of 111-2 (yield 5.6 g, 72% ) . [ (Y]D-40.0" (c, 0.422 in MeOH) . Anal. Calcd. for C33H54N608S 0.5H20: c , 56.31; H, 7.88; N, 11.94. Found: C, 56.37; H, 7.80; N, 11.91.

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Boc-Leu-Leu- Thr (621)-Cys (Acm) -Val-NHZ (1115). Fragment 111-5 was also synthesized by the same procedure as described for the synthesis of 111-2 (yield 4.96 g, 93% ). [ a]D -5.3" (c, 0.260 in D M F ) . Anal. F o u n d C, 57.66; H, 8.10; N, 12.00. Calcd. for C39H65N70gS * 0.2H20: C, 57.71; H, 8.12; N, 12.08. F o u n d C, 57.66; H, 8.10; N, 12.00. Boc-Pro-Leu-Leu- Thr (621)-Cys (Acm)- Val-NH2 (1116). Fragment 111-6 was also synthesized by the same procedure a s described for the synthesis of 111-2 (yield 3.8 g, 85%) . [ a]D -25.6" (c, 1.081 in D M F ) . Anal. Calcd. for C44H,2N801&3 0.7H20: C, 57.58; H, 8.06; N, 12.21. F o u n d C, 57.59; H, 8.03; N, 12.15.

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Boc-Ala-Cys (Acm) -Ser (Bzl) - Thr (Bzl) -Cys (Acm)Ala-Gly-OH (11-7-OH). A solution of 11-7 (500 mg, 0.43 mmol) in AcOH (30 m L ) containing Zn powder (560 mg, 8.6 mmol) was stirred for 2.5 h a t 40°C. After the reaction, the reaction mixture was filtered. T h e filtrate was concentrated a n d ether was added to give white powder.

SYNTHETIC ANALOGUE FOR PLANT-TYPE FERREDOXIN

Boc-Ala-Cys (Acm) -Ser (Bzl) -Thr (Bzl) -Cys (Acm) Ala-Cly- Pro4 eu-leu- Thr (Bzl)-Cys (Acm)-Val-NH2 (11-111). Fragment 11-111was synthesized from frag-

ments 11-7-OH and deblocked 111-6 by the following method. Fragment 111-6 (360 mg, 0.4 mmol) was dissolved in TFA ( 3 m L ) a t -6°C. T h e reaction mixture was concentrated to a half volume under reduced pressure, 4.8 N HCl/dioxane (0.16 mL, 1 mmol) was added to the solution. All solvents were evaporated in vacuo. T h e residue was washed with ether, dried over NaOH pellets in vacuo. The residue was dissolved in DMF (50 mL) together with HOBt (60 mg, 0.44 mmol) and 11-7-OH (0.43 mmol). WSCI (0.08 mL, 0.44 mmol) was added t o the stirring solution a t -20°C. After the reaction, the solution was concentrated under reduced pressure. The crude product was triturated with H 2 0 , 1/ 2 N HCl aq., water, 5% NaHC03 aq. and water, successively and followed by precipitation from MeOH/CHC13 / diethyl ether t o give white powder (440 mg, 60% ) . [.ID -21" (c, 0.318 in DMF). Anal. Calcd. for CS5H129N17021S3 * 2.9H20: C, 54.49; H, 7.25; N, 12.71. F o u n d C, 54.48; H, 7.19; N, 12.57. Ac

- Pro - Tyr[Bz(Cl)2]- Ser(Bz1) - Cys(Acm) - Arg- Gly - Ala - Cys(Acm) - Ser(Bz1) - Thr-

(Tos) - Ala

(Bz1)-Cys(Acm)-Ala-Gly-Pro-Leu-Leu- Thr(Bz1)-Cys(Acm) - Val - NH, (Blocked-20-Peptide). Fragment 11-111 (400 mg, 0.22 mmol) was dissolved in TFA ( 3 m L ) at -6°C. T h e reaction mixture was concentrated t o about a half volume under reduced pressure, 4.8 N HCl/dioxane (0.086 mL, 0.41 mmol) was added t o the solution. All solvents were evaporated in vacuo. T h e residue was washed with ether, dried over NaOH pellets in vacuo. T h e residue was dissolved in DMF (30 m L ) together with HOBt (33 mg, 0.24 mmol) and Ac-1-7 (300 mg, 0.24 mmol). WSCI (0.044 mL, 0.24 mmol) was added to the stirring solution at -20°C and after the reaction, the solution was concentrated under reduced pressure. The residue was triturated with HzO and washed with MeOH.

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Ac - Pro - Tyr - Ser - Cys(Acm) - Arg - Ala Gly - Ala Cys(Acm)- Ser Thr- Cys(Acm)- A la - Gly - Pro - L eu - L eu - Thr - Cys(Acm) - Val - NH, [Cys(Acm) - 20 - Peptide]. Blocked-20-peptide (230 mg, 0.08 mmol) was treated with HF and anisole (0.5 mL, 4.6 mmol) by

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the literature method.13 T h e solution was applied t o CM sephadex column by elution with AcOH-AcONH, buffer ( p H 5 ) solution with linear gradient from 0.02 to 0.1M. Fractions with AZa0were applied t o sephadex G-25 and eluted by a n AcOH-NH40Ac buffer ( p H 5 ) solution with a linear gradient from

1539

0.02 to 0.1 M . T h e fractions were purified by LH-20 column with a n AcOH-H20 0.1% solution. T h e purified fractions were concentrated under reduced pressure t o give white solid (34 mg, 6.7% ) . Cys-20-pepfide (20-Pep). T o a n aqueous solution

( 5 m L ) of Cys ( Acm) -20-peptide ( 10 mg, 4.4 X mol) was added H,C12 (7.2 mg, 2.5 X lop5 mol) at room temperature. The solution was stirred vigorously for 4 h and washed three times with 6 mL of diethyl ether. The solution was flushed with argon gas, followed by blowing hydrogen sulfide through for 3 h a t room temperature. Black precipitate was removed off by filtration. The filtrate was lyophilized and the white residue was kept under argon atmosphere. Synthesis of [ FezS,(20-Pep)]. All manipulations were carried out under argon atmosphere. Two methods were used for the synthesis of [ Fe2S2( 20p e p ) ] . Method ( a ) is by the reaction of Cys-20-peptide and [FezS2C14]2pwith the addition of triethylamine in DMF ( 5 m L ) .I4-l6Method ( b ) is by a ligand exchange method between the Cys-20-peptide and ( NEt4)J Fe2S2(S - ~ - B U )in~ DMF.8 ] The ligand exchange method is more suitable for the quantitative formation of 20-peptide/Fe2S;' complex in a DMF solution because t-butanethiol liberated was easily removed in vacuo. Method ( b ) is a s follows: a DMF solution of Cys20-peptide (4.4 mg, 2.2 X lop6mol) was mixed with a DMF solution ( 3 mL) of [NEt4]2[Fe2S2(S-t-Bu)4] mol) a t room temperature. The (1.5 mg, 2.2 X solution was concentrated under reduced pressure a t room temperature. The residue was dissolved in 2.5 mL of DMF to prepare the solution (concentration, 3.7 x H - ~ M ) .

Physical Measurements

Absorption spectra were measured on a JASCO UVIDEC-5A spectrophotometer in visible region. CD spectra were recorded on a JASCO 5-40 spectropolarimeter. A cell of 1-mm cell path length was used for the absorption and the CD spectral measurements. T h e t and At values were calculated in units of molpl cmp'. T h e 400-MHz 'H-nmr spectra were recorded on a JEOL GSX-400 spectrometers at 30°C for GX-400. Spinach ferredoxin (10 mg) in 5 mL of 3 M NaCl aq. were dialyzed five times with D 2 0 for desalting a n d finally 0.5 mL of the solution was taken. T h e solution was added t o 0.5 mL Me,SO-d, t o measure the 'H-nmr spectrum of the denatured ferredoxin a t room temperature. ESR

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UEYAMA ET AL.

spectra were obtained a t 77 K on a JOEL JES FEIX instrument with 100 kHz magnetic field modulation. T h e g value was standardized using the 3,3,7,7,-tetracyanoquinodimethane radical (g = 2.0025) and Mn (11) (g = 1.981 ) . T h e ESR sample was prepared by the following method. One milliliter solution (8.7 X 10-4M) of [ NEt4I2[Fe2S2(20-pep)l was mixed with 0.2 mL of DMF solution (3.7 X 10-4M) of sodium dithionite a t room temperature. Differential pulse polarograms were performed under argon on a YANACO P-1100 polarographic analyzer. Sample solutions were 2 X lop4mol dm-3. [ N (rz-Bu),] C10, and potassium chloride were used as a supporting electrolyte. The voltammograms were recorded a t room temperature with a SCE as the reference.

RESULTS AND DISCUSSION Synthesis

Two underscored fragments in Figure 2 correspond to the Pro (38)- Gly(51) and Leu (77) - Val (80) around the active site of S.platensis ferredoxin. The remaining fragment, Pro-Leu, which was employed instead of T h r ( 52) - Val( 76) in the native ferredoxin, was expected to form a P-turn preferably. The synthesis of Cys ( Acm) -20-pep was accomplished by the sequential condensation in solution between Pro -Gly, Ala -Gly, a n d P r o -Val fragments. The blocked Cys ( Acm) -20-peptide was isolated by chromatographic purification with Sephadex G-25. The purity of Cys (Acm) -20-pep was examined by observation of four sets of 'H-nmr signals of Cys ( Acm) methyl groups at 1.84,1.85, 1.86, and 1.88 ppm in D20. Direct synthesis of the complex 1 was attempted from the reaction of FeC13, S2-, and the deblocked Cys-20-peptide in a n aqueous solution. The reaction mixture gave a n absorption maximum a t 400 nm due t o the (Fe,S,) *+-typecomplex t h a t gradually decomposed by hydrolysis and decolorized. The formation of the ( Fe4S4)'+ core is consistent with the facile conversion from [ 2Fe-2S ] complex to [4Fe-4S] complex in the presence of protic medium, e.g., alcohol or water.l4>l5 ( NEt4)2[Fe&( 20-pep)] ( 1 )was synthesized by methods ( a ) and ( b ) (see Experimental). Unavoidable contamination of chloro-derivatives [Fez&C14]2- was encountered in method ( a ) since the reported Fe-S bond strength (81 & 5 kcal mol-') is close to Fe-C1 bond strength (ca. 84 kcal mol-l) .17 We successfully synthesized 1 by method ( b ) . Complex 1 is sensitive t o air or water, soluble in DMF or dimethyl sulfoxide, and insoluble in acetonitrile and dichloromethane.

-

-

Visible and CD Spectra of [FezSz(20-Pep)l

A DMF solution of [Fe2S2(20-pep)]( 1 ) exhibits characteristic ligand-to-metal charge transfar absorption maxima a t 423 nm ( t : 6580) and 461 n m ( E: 5000), which are to be compared with the maxima a t 423 nm ( E : 9700) and 466 nm ( t : 8520) for native ~ absorption oxidized [ 2Fe-2SI f e r r e d ~ x i n .The maxima are also similar to those of alkanethiolate [ 2Fe-2SI model complexes, [ FezSz( S2-o-xyl)z] and [ Fe2S2( Z-~ys-Ala-Ala-cys-OMe)~] '-, which have been reported to show the absorption maxima a t 414 n m ( E : 11,000) and ca 455 nm ( t: 9200)6 and a t 417 n m ( t : 10,000) and 450 nm ( t : 8900) respectively, in DMF. T h e addition of water to a DMF solution of 1 resulted in decomposition concomitantly with disappearance of the absorption maxima a t 423 and 461 nm. T h e results explain the previous failure of the direct synthesis of 1 from FeCl,, S '-, and 20pep in aqueous solution. Therefore, it is likely that the peptide chain does not form a favorable conformation to wrap around a n ( Fe2S2)'+ core with sufficient protection from water. The CD extrema of 1 in DMF are observed a t 380 (At: -0.1) , 396 (At: +0.2), 413 ( At: -0.2), 458 ( AE:+0.4), 490 ( A t : +0.1), and 592 ( At: -0.3). The denatured form of spinach ferredoxin in 30% DMF/ H 2 0 exhibits CD extrema a t 380 ( Ac: -3.8), 398 ( AE:-0.6), 410 ( A t : -3.6), 470 ( A t : +3.8), and 550 nm (At: -1.6). Thus, the synthetic complex 1 shows weak CD extrema in the whole visible region presumably due to the compensation of two different sets of CD extrema by the mixture of two coordination isomers as described later. The three transition wavelengths a t 350 nm 500 nm for 1 are similar t o those for the denatured form of planttype ferredoxin, although the native protein is known to give a large extremum a t 470 nm.I8 T h e results suggest that a t least one of the isomers has almost the same local structure as the active site of denatured plant-type ferredoxin. In a n aqueous Triton X-100 solution, plant-type ferredoxin was reported to give a denatured form.I8 The two characteristic absorption maxima of 1 in 10% aqueous Triton X-100 solution were also observed in the same region as those of plant-type ferredoxin reported in DMF. T h e present results suggest the presence of a stable [2Fe-2S] core in hydrophobic part of micelles. T h e 10% aqueous Triton X-100 solution allows the formation of large micelles over the CMC point. If 1 has a similar structure to that of native plant-type ferredoxin, the hydrophobic amino acid side chains of the 20-pep ligand are di-

'-

-

SYNTHETIC ANALOGUE FOR PLANT-TYPE FERREDOXIN

rected outside on exposure to bulk water in micelles and protect the ( Fe2S2)" core from hydrolysis. Electrochemical Properties

The redox potential of 1 in solution was obtained using pulse polarography and cyclic voltammography. A DMF solution of 1 exhibited two distinct redox couples a t -0.64 V vs SCE = 0.94) and -0.96 V = 0.95) for (Fe2S,)2'/(Fe,S,)+ a t room temperature as shown in Figure 3. It is apparent that a tridentate peptide fragment ( Cys-ArgAla-Gly-Ala-Cys-Ser-Thr-Cys) in 1 chelates to a ( Fe2S,) core with the following two coordination modes and results in giving two isomers (a and b ) as shown in Figure 4. Isomer a possesses two Cys thiolate ligands of the Cys-Ser-Thr-Cys fragment bridging between the two Fe( 111)ions. Then the CysArg-Ala-Gly-Ala-Cys fragment chelates to one of the two Fe(II1) ions. The other structure b has the bridging fragment of Cys-Arg-Ala-Gly-Ala-Cys and the chelating Cys-Ser-Thr-Cys fragment t o one Fe( 111) ion. The electrochemical results indicate the formation of the two isomers in a ca. 1 : 1 ratio consistent with the results by the 'H-nmr analysis of Cys CH2 signals. Simple [2Fe-2S] ferredoxin model complexes having alkane- or arenethiolate ligand have been reported to exhibit a negative redox

(a,/&,

(&,/a,

'+

1541

potential in DMF, e.g., -1.49, -1.09, and -1.31 V vs SCE for [FezS,(S2-o-xyl),lz-, [Fe2S,(SPh),I2-, and [ Fe2S2( S-t-Bu),] 2-.5,6*R The difference in the redox potential between the two isomers is probably caused by the formation of NH -S hydrogen bonds that mainly contribute to the positive shift of redox potential in rubredoxin and [ 4Fe-4SI ferredoxin model c o m p l e ~ e s . ' ~ Further ~ z ~ study to elucidate the specific position of the hydrogen bonds will be required. Denatured spinach ferredoxin in H,O/DMF ( v / v 6 / 4 ) exhibited a redox potential at -0.69 V vs SCE, whereas the native spinach ferredoxin showed the couple a t -0.64 vs SCE in a n aqueous solution. T h e redox potential of the couple in 1 a t -0.64 V vs SCE in DMF was almost the same as those of native and denatured spinach ferredoxins in aqueous and DMF (40%) solutions, respectively, and was tentatively assigned as that of isomer a. Thus, the positive-shifted redox potential of isomer a in 1 in organic solvent has a n advantage for the reduction by a mild reductant including biologically important ones such as NADPH. The redox couple a t -0.96 V vs SCE is assigned to that of isomer b, since [ Fe2S2( Z - c y s - X - Y - ~ y s - O M e )2p~ ](X-Y = Ala-Ala, Thr-Val) exhibits a redox couple at -1.06 -1.09 V vs SCE (i,,,/i,,, = 0.8). T h e electrochemical results indicate that isomer a of [ Fe2S2( 20-pep)]'-has a similar environment as the ( Fe,S,) core site of the native and the denatured ones. Although it is not possible to examine the electrochemical properties of spinach ferredoxin in low dielectric solvent, e.g., dichloromethane, because of its insolubility, the realization of the same redox potential (-0.64 V vs SCE) by a n artificial model complex 1 as that of native spinach ferredoxin suggests that the Cys-20-peptide ligand ranges almost all over the (Fe2S,) core. The hydrolytic stability observed also indicates very hindered access of the solvent t o the (Fe2S,)" core.

-

'+

-0.96

'+

'H-NMR Spectra of [Fe2S2(20-Pep)]and Denatured Plant-Type Ferredoxin

-0.5

-1 .o

V (vs SCE) Figure 3. Differential pulse polarogram of (NEt,),[ Fe2S2( 20-pep ) ] in DMF a t room temperature.

'H-nmr spectra of denatured spinach ferredoxin in D20-Me2SO-d6 ( v / v , 1/ 1) mixed solvent and ( NEt4)2[Fe,S,( 20-pep)] ( 1 ) in MezS0-d~and in the Cys CH, region are shown in Figure 5. The mixed solvent is reported t o disrupt the folding of native spinach ferredoxin.I8 The denatured plant-type ferredoxin exhibits two peaks of Cys CH, a t 39.0 and 32.3 ppm due t o the contact-shift through Cys thiolate from the (Fe2S2) core and these signals exhibit anti-Curie behavior with the change of temperature.

'+

1542

UEYAMA ET AL.

T

NH2

Isomer a

-4 -3t- B uSH

AC

Isomer b Figure 4. Two possible chelations (isomers a and b ) by the 20-pep ligand to an (Fe,S,) '+ core. Isomer a is isostructural to a part in the structure of native ferredoxin reported crystallographically.12The circle refers to amino acid residue except Cys residue. SH groups are supplied from the Cys residues.

Similar contact-shifted,broad signals a t 13 ppm and 20-40 ppm has been observed for spinach, parsley, and vertebrate ferredoxins, and assigned to Cys CH, nuclei of the Cys ligands that were established by nuclear Overhauser effect studies of plant-type ferredoxin.2"26 Recent 600-MHz spectra of vertebrate ferredoxins have shown one broad signal overlapped with several broad signals in the region of 25-40 ppm at ambient t e m p e r a t ~ r e . 'The ~ observation of the two distinct separate Cys CHZ signals for denatured spinach [2Fe-2S] ferredoxin is due to the (FezS2)2 f core environments different from that of the native ferredoxin. T h e difference is coincident with that in the CD results for the both forms of spinach [ 2Fe-2SI ferredoxin. The 20-peptide complex 1 in Me2SO-d, exhibits three peaks of the Cys CH2 group a t 39.7, 33.9 and 24.5 ppm. The two low-field peaks at 39.7 and 33.9

ppm appear in the same positions of the two Cys CHz signals of denatured spinach ferredoxin. T h e four Cys residues of 1 are thus found to exist under similar environments as those of denatured spinach ferredoxin. Previously, we reported that [Fe2S2(Z-cys-AlaAla-cys-OMe),] containing a ( S , S )-chelating peptide ligand exhibits two separate Cys CH2signals at 30.7 and 22.9 ppm, and that the tetrapeptide preferably chelates to one Fe( 111)ion rather than bridge the two Fe(II1) ions: A low field peak a t 24.5 ppm for 1, accompanied by a peak a t 30 34 ppm, shows the presence of another isomeric [ ZFe-ZS] complex with structure b (Figure 4) in solution. Isomer a is isostructural t o the active site of S. platensis ferredoxin." T h e signal intensities indicate the presence of the two isomers in ca 1 : 1 ratio. It is likely that the presence of the two isomers is due to the equal

'-

-

SYNTHETIC ANALOGUE FOR PLANT-TYPE FERREDOXIN

16.4

4

39.0

i

(Fez&) complex in the solution since only isomer a having a positive-shifted redox potential (-0.64 V vs SCE in D M F ) can be reducible by the mild reductant. T h e spectrum is quite similar t o denatured ferredoxins reported for Halobacterium halobium ferredoxin a n d for Spirulina maxima ferredoxin." Simple [ 2Fe-2SI model complexes exhibit a n E S R signal with g = 2.014, 2.000, and 1.963 for [Fe2Sz(S-t-Bu)4]2-or with g = 2.014, 1.982, and 1.959 for [ FezSz( Z-cys-Ala-Ala-cys-OMe)z] 2p in DMF at 77 K. T h e addition of a n excess of the reducing reagent resulted in decomposition of the (Fe2Sz)+species exhibiting a signal at g,, = 4.26 and 4.55. Detailed ESR studies of various synthetic simple thiolate complexes have been done by Beardwood and Gibson," who compared the ESR results with t h a t of native [ 2Fe-2SI ferredoxins. They discussed the values ofgy-gx,which are considered to be related with a rhombic Cpvdistortion in the ligand environment of Fe ion.30In the case of plant-type [ 2Fe-2SI ferredoxin, a decrease of the large g,-g, splitting by addition of a denaturing reagent has been reported.31 T h e large gy-gx value (74 cm-l) of reduced 1 presumably reflects a distortion of the (Fe2S2)+core even in the denatured model complex. T h e results suggest that each Fe ion in form a is surrounded with a different environment formed by the Cys-AB-C-D-Cys-X-Y-Cys chelation, which induces the partial formation of NH -S hydrogen bonds around one of the two Fe ions. Actually, the crystallographic analysis of S. platensis [ 2Fe-2SI ferredoxin demonstrated that one of the two Fe ions partially has more NH-S hydrogen bonds different from another Fe ion." As described above, one (form a) of the isomers has similar [ 2Fe-2SI core environments to those in the metal site of the denatured ferredoxins relaxed from the polypeptide constraints. T h e similarity in redox potentials between 1 and native or denatured [2Fe-2S] ferredoxin is due to the presence of NH -S hydrogen bonds formed by the synthetic oligopeptide or the protein polypeptide chain. +

32.3

33.9

1543

15.0

I

I

I

I

40

30

20

10

GrPPm

Figure 5 . The 400-MHz 'H-nmr spectra of ( a ) the Cys CH2 region of denatured spinach ferredoxin in DzO/ Me,SO-d, ( v / v 1/ 1) solution and ( b ) ( NEt412[ Fe2S2( 20pep)] in Me,SO-d,.

chelating abilities of both peptide fragments to one of the two Fe(II1) ions in DMF, which is a strong solvating medium against the peptide chain. Similar two separate Cys HAHBsignals have been found for reduced rubredoxin model peptide complexes having chelating Cys-X-Y-Cys ligands. For example, the preferable chelation of a Cys-Pro-Leu-Cys fragment to one Fe( 11) ion gives two separate Cys CH, signals a t 150-200 and 220-250 ppm due to the diastereotopic ABX system.28 A relatively sharp signal appears at 16 ppm with a still significant contact shift for the denatured plant-type ferredoxin and a t 15 ppm for 1. T h e above peak observed in 10 20-ppm region is assignable to Cys CaH signal for the denatured ferredoxin since similar signals in the region have been reported to be observed for native plant-type ferr e d o ~ i n s . ' " Ho ~ ~wever, ~ ~ ~ there is a possibility that [ 4Fe-4SI -type ferredoxin and [ 4Fe-4SI peptide complexes generated from the corresponding [ 2Fe2S] complexes or denatured [ 2Fe-2SI ferredoxin exhibit similar Cys CH2 signals in this region (10 15 ppm).7

-

-

Conclusions

ESR Spectra of the Reduced Model Complex The formation of a characteristic [ 2Fe-2SI core is also confirmed by the ESR signals by detection of the ( Fe2S2)+species in the reduced 1. T h e reduced (Fez&) species of 1 by addition of a n aqueous solution of 18-crown-6 * NazSz04exhibits a n ESR signal with g, = 2.014, g, = 1.973, and g, = 1.899 a t 77 K. T h e spectrum indicates the presence of one +

T h e artificial construction of the chemical environments of the [2Fe-2S] active site using the oligopeptide fragment ( 20-pep) realizes a positive shift of redox potential (-0.64 V vs. SCE in D M F ) as t h a t (-0.64 V vs SCE in as.) of plant-type ferredoxin, different from those of the simple [ 2Fe-2SI alkanethiolate complexes. The peptide circumstance around the ( Fe2Sz) core of the model complex is

'+

1544

UEYAMA E T AL.

thus similar to that in the metal site of the denatured form of plant-type ferredoxin. Therefore, upon incorporation of an (FeeS2)'+ core into 20-pep in DMF, the Fe-S bond formation occurs randomly to give at least two coordination isomers. In the case of the denatured form of the plant-type ferredoxin, the original Fe-S (cys) bonds are maintained even with the random conformation of the surrounding protein. Our results strongly suggest the importance of a characteristic protein conformation near the Cys residues to specifically incorporate the (Fees2)'+ core.

REFERENCES 1. Masaki, R., Yoshikawa, S. & Matsubara, H. ( 1982) Biochem. Biophys. Acta 700, 101. 2. Tagawa, K. & Arnon, D. I. (1968) Biochem. Biophys. Acta, 153, 166. 3. Estsbrook, R. W., Suzuki, K., Mason, J. I., Baron, J., Taylor, W. E., Simpson, E. R., Purvis, J . & McCarthy, J. ( 1973) Iron-Sulfur Proteins, Vol. 1, Lovenberg, W., Ed., Academic Press, New York, chap. 3. 4. Cammack, R., Rao, K. K., Bargeron, C. P., Hutson, K. G., Andrew, P. W. & Rogers, L. J. (1977) Biochem. J. 168,205. 5. Mayerle, J. J., Frankel, R. B., Holm, R. H., Ibers, J. A., Phillips, W. D. & Weiher, J. F. (1973) Proc. Natl. Acad. Sci. U S A 70, 249. 6. Mayerle, J. J., Denmark, B. V., DePamphilis, B. V., Ibers, J. A. & Holm, R. H. (1975) J . A m . Chem. SOC. 97, 1032. 7. Balasubramaniam, A. & Coucouvanis, D. (1986) Inorg. Chim. Acta 78, L35. 8. Ueno, S., Ueyama, N., Nakamura, A. & Tsukihara, T. (1986) Inorg. Chem. 25, 1000. 9. Stephens, P. J., Thomson, A. J., Dunn, J. B. R., Keiderling, T. A., Rawlings, J., Rao, K. K. & Hall, D. 0. (1978) Biochemistry 17,4770. 10. Ueyama, N., Ueno, S. & Nakamura, A., (1987) Bull. Chem. SOC.Jpn. 60, 283. 11. Tsukihara, T., Kobayashi, M., Nakamura, M., Katsube, Y., Fukuyama, K., Hase, T., Wada, K. & Matsubara, H. (1982) BioSystems 15, 243. 12. Fukuyama, K., Hase, T., Matsumoto, S., Tsukihara,

T., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K. & Matsubara, H. (1980) Nature 286, 552. 13. Sakakibara, S., Shimonisi, Y., Okada, M., Sugihara, H. (1967) Bull. Chem. SOC.Jpn. 40, 2164. 14. Camberay, J., Lane, R. W., Wedd, A. G., Johnson, R. W. & Holm, R. H., (1977) Inorg. Chem. 16,2565. 15. Do, Y., Simhon, E. D. & Holm, R. H. (1983) Inorg. Chem. 22, 3809. 16. Gaydon, A. G. (1974) Dossociation Energies and Spectra of Diatomic Molecules, 3rd ed., Chapman and Hall, London. 17. Kondratiev, V. N. ( 1974) Bond Dissociation Energies, Ionization Potentials and Electron Affinities, Mauka Publishing House, Moscow. 18. Bonomi, F. & Kurtz, P. M., Jr. ( 1982) Biochemistry 2 1,6838. 19. W.-Y. Sun, W.-Y., Ueyama, N. & Nakamura, A. (1991) Inorg. Chem. 30,4027. 20. Ueyama, N., Nakata, M., Fuji, M., Terakawa, T. & Nakamura, A. (1985) Inorg. Chem. 24, 2190. 21. Ueyama, N., Terakawa, T., Nakata, M. & Nakamura, A. (1983) J . Am. Chem. SOC.105, 7098. 22. Ohno, R., Ueyama, N. & Nakamura, A. (1991) Inorg. Chem. 30, 4887. 23. Dugad, L. B., La Mar, G. N., Banci, L. & Bertini, I. (1990) Biochemistry 29, 2263. 24. a ) Skjeldal, L., W. M. Westler, W. M. & Markley, J. L. (1990) Arch. Biochem. Biophs. 278,482. 25. Skjeldal, L., Markley, J. L., Coghlan, V. M. & Vickery, L. E. (1991) Biochemistry 30,9078. 26. Skjeldal, L., Westler, W. M., Oh, B.-H., Krezel, A. M., Holden, H. M., Jacobson, B. L., Rayment, I. & Markley, J. L. (1991) Biochemistry 30, 7363. 27. Skjeldal, L., Markley, J. L., Coghlan, V. M. & Vickery, L. E. (1991) Biochemistry 30,9078. 28. Bertrand, P. & Gayda, J. P. (1979) Biochem. Biophys. Acta 579, 107. 29. Beardwood, P. & Gibson, J. F. (1983 ) J . Chem. SOC. Dalton Trans. 738. 30. Bertrand, P. & Gayda, J. P. ( 1980)Biochern. Biophys. Acta 625, 337. 31. Cammack, R. (1975) Biochem. SOC.Trans. 3,482.

Received January 21, 1992 Accepted April 29, 1992