Superoxide dismutase activity of macrocyclic polyamine complexes

172

Biochimiea et Biophysica .4 eta, 678 ( 1981) 172 - 179

Elsevier/North-HollandBiomedicalPress BBA 29777 SUPEROXIDE DISMUTASE ACTIVITY OF MACROCYCLIC POLYAMINE COMPLEXES EIICHI KIMURA,ATSUKOSAKONAKAand MIEKONAKAMOTO Department of Medicinal Chemistry, Hiroshima University School of Medicine, Kasumi 1-2-3, Minami-Ku, Hiroshima, 734 {Japan}

(Received May 4th, 1981)

Key words: Polyamine derivative; Superoxide dismutase; Cyclic compound

Copper(ll) and nickel(ll) complexes of macrocyclic polyamine derivatives possessing partial oligopeptide-like structures are found to suppress the xanthine-xanthine oxidase-mediated reduction of nitroblue tetrazolium and also to suppress formazan formation by potassium superoxide. The activity in the superoxide dismutase assay is dependent on ring size, type and number of donor atoms, metal ion, and substituents on the macrocycles. Some of those are more active than the known O~ scavengers such as eopper(ll)-salicylate and copper(ll)-amino acid (or peptide) complexes. Nickel (ll)-naphthylmethyldioxo-[16]ane Ns, 13, 1 : 1 complex (NiH_:L) is the most active among the 30 chelates examined.

Introduction Superoxide dismutase, an enzyme catalyzing the reaction 20~ + 2 H+ ~ 02 + H202, constitutes an important defense system against oxygen toxicity [1 ] in living organisms. The mammalian enzyme has a copper atom at the active site. Recently, activated oxygen species such as O~ and "OH have been implicated in the initiation of inflammatory types of arthritis [2], which suggest that the O~ scavengers may be useful in repressing arthritis. There have been some attempts to use superoxide dismutase for the treatment of arthritis [3]. Sorenson [4] found that many antiarthritic drugs, such as aspirin, are more active against inflammation in the form of Cu(II) complexes than in their free forms, suggesting that the in vivoformed Cu(II) complexes may be responsible for their therapeutic action. Diminished amounts of superoxide dismutase have been found in some tumors [5]. The lower levels of superoxide dismutase would permit an increase in amount of the toxic O~ causing various biochemical disorders and, ultimately, the characteristic biological phenomena of cancer cells. Therefore, a search for molecules possessing the superoxide dismutase activ-

ity might offer a step forward to exploration of cancer therapy [8]. Some Cu(II) complexes that exhibit the superoxide dismutase activity have already proved themselves to be antitumor agents [9,10,11 ]. In a series of investigations to screen macrocyclic polyamines for biochemical and physiological tests [12,13], we have examined the superoxide dismutase activity of their copper(II) and nickel(II) complexes of various types of macrocyclic polyamines (Fig. 1). Very recently [14-16], we discovered that macrocyclic dioxotetraamine ligands form stable Cu(II) and Ni(II) complexes. These demonstrated striking similarities to oligopeptide complexes both chemically and physicochemically. In light of the fact that a number of amino acid and peptide complexes of Cu(II) possess superoxide dismutase activity [1724], the dioxopolyamine macrocyclic system might also be active in the superoxide dismutation. Using the xanthine-xanthine oxidase assay and KO2 assay, we have found that some possess strong superoxide dismutase activity. A few of them are more active than the previously known copper(II), amino acid or salicylate complexes. The activities are discussed in terms of the macrocyclic ring size, number of N donor atoms, metal ion and substituents on the basic skeletons, factors determining the struc-

0304-4165/81/0000-0000/$02.50 © 1981 Elsevier/North-Holland BiomedicalPress

173 R

R

0

H::~"~H NH

H~N.~

~H

N~

H~

H.~. H

2

oN ~ - o

H

H

HNX,__/N

R=H

4 O~/""S""~ O ' HN NH

15

19

3

7

HN

o< "Zo

o ,,o

""

?

17

20

21.

HN~--~NHk~ HNI"A~IN H.H

22

NH

10

16

23

H

R

0

N N

L.~HN..~

~ N _/N?

9

8 R=(CH2)3CH 3

0

O~,'..NH -~X~.O

N~

6 R=H

R=CH2~

0

H~'~ H

~

Nv~

H 1

O

0

R=H

11 12

R=CH20

13

R=CH2~

14

R=(CH2)3CH 3

18

24

H~NN~'~I NN~~ H HN/~ H

25

26

H /~OH

H,

H 27

28

29

H2 30

31

CH2CO2H

32

Fig. 1. Structural formulae of the polyamine and the relevant ligands used for the present study.

tural and electronic properties of the metal complexes.

Experimental Materials

The macrocyclic polyamines tested were all synthesized, and identified with elemental analysis and other suitable analytical techniques developed ~ this laboratory [25]. Bovine superoxide dismutase and xanthine oxidase were purchased from Sigma, and potassium superoxide (KO2) from Alfa. Dirnethyl sulfoxide (DMSO) was redistilled over molecular sieves. All the other chemicals used were of analytical grade. The stock solutions of the macrocyclic chelates (all 1 : 1 complexes except for 2 : 1 for M-17 and -18), were prepared by mixing the calculated amount of 0.1 M CuCI2 or 0.1 M NiC12 with 10% excess ligand in 0.05 M sodium carbonate buffer (pH 10.2).

Under these conditions, the metal ions are practically 100% complexed with any of the macrocyclic polyamines, as shown in our previous studies [14-16,26, 27]. The stock solutions of Cu(II) chelates of amino acids (2 : 1 complexes) and oligopeptides (1 : 1) complexes) were also prepared by mixing 0.1 M CuC12 with 10% excess ligands in 0.05 M sodium carbonate buffer. Superoxide was generated by the xanthinexanthine oxidase system [28] and by the potassium superoxide system [29]. Measurements

The superoxide dismutase activity was first examined by a modified method of the xanthine-xanthine oxidase system of Imanari et al. [30]. Aqueous solutions of 3 mM xanthine, 4 mM ethylenediamine diaeetate, 0.15% bovine serum albumin and 0.75 mM nitroblue tetrazolium (NBT) (0.1 ml of each)were mixed into 2.4 ml of 0.05 M sodium carbonate buffer (pH 10.2). The macrocyclic chelate solution at vary-

174 TABLE I THE SUPEROXIDE DISMUTASE ACTIVITY OF COPPER AND NICKEL COMPLEXES OF MACROCYCLIC POLYAMINES AND THE RELEVANT COMPOUNDS E, redox potential for M(II) ~ M(III) in aqueous solution at pH ~ 10. L, ligand. Complex No.

Complex formulae

Concentration of complex required to yield 50% inh~ition (#M) Xanthine-xanthine oxidase system

Superoxide dismutase (bovine) Cu(II) complex 1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 glycine glycylglycine 31 32 histidine lysine glycylglycyl histidine Salicylate Phenanthroline Ni(II) complex 1 2 3

0.04 Cu(H_2L) a Cu(H_2L) a Cu(H_2L) a Cu(H_2L) b Cu(H_2L) b Cu(H_2 L) a Cu(H_2L) b Cu(H_2L) b Cu(H_2L) b Cu(H_2L) b Cu(H_2L) b Cu(H_2L) b Cu(H_2L) b Cu(H_2 L) b Cu(H_2L) b Cu2 (H..4 L) Cu2(H..4L) CuLc CuL d CuLc CuL(OH) e CuLc CuL c CuLc CuL c CuL d CuL c CuLc CuL b CuL2 Cu(H_ 1 L) Cu(H_2L) a

>2 >2 >2 >2

>2 >2 >2

>2

>2 >2 >2 >2 >2 >2 >2 >2

Cu(H-aL) a

500 000 000 000 000 400 400 800 400 900 000 000 000 300 500 200 220 000 600 500 600 000 000 000 000 000 000 000 000 600 700 400 400 700 600 000 400 200

CuL2 CuL2 Cu(H- 2 L) CuL2 CuL2

>2

Ni(H_2L) a Ni(H_ 2 L) a Ni(H_2L) a

>2 000 >2 000 >2 000

E (V vs. standard Calomel electrode)

K O 2 system

0.2 200

0,42 a 0.56 a 0.64 a

0,66 b 300 800

500

0.69 a

0.93 0.94 O.68 0.74 0.77

b b b b b

0.85 b

200

0.67 a 0.38 a

400 > 2 000 90 0.62 a 0.90 a 0.80 a

175 TABLE I (continued) Complex No.

Complex formulae

Concentration of complex required to yield 50% inhibition (~M) Xanthine-xanthine oxidase system

6 10 ll 12

13 14 20 27 29

Ni(H~L) a Ni(H_2L) b Ni(H-2L) b Ni(H_2L) b Ni(H-2 L) b Ni(H_2L) b NiL d NiL d NiL b

>2 000 >2 000 200 30 9 >2 000 >2 000 >2 000 >2 000

E (V vs. standard Calomel electrode)

KO2

0.62 a 200 50 20

0.24 b 0.24 b 0.25 b

a See Ref. 16. b Unpublished data. c See Ref. 27. d Complex formula is not firmly established under the given conditions, hence a tentative complex stoichiometry is given. e See Ref. 43.

ing concentration was added and the resulting mixture was allowed to stand at 25°C for 10 min. An aqueous solution of xanthine oxidase (0.1 ml)was then added to initiate the reaction. After exactly 20 min, the reaction was quenched by adding 0.1 ml of 6 mM CuC12. The absorbance at 560 nm resulting from the reduction of rtitroblue tetrazolium was measured. The macrocyclic chelates used as superoxide dismutation agents inhibited this reaction to diminish the absorbance at 560 nm. The amount of xanthine oxidase to be added was adjusted so as to yield the absorbance of 0.20-0.23 in the absence of the metal chelates. The present assay method was applied to measure the activity of the commercial bovine superoxide dismutase, the known superoxide dismutase model complexes, and the macrocyclic polyamine complexes simultaneously. To check uncontrolled side reactions of the components required in the xanthine-xanthine oxidase system, a strictly inorganic potassium superoxide system was employed. The absorption of the formazan color at 560 nm following the addition of 0.1 ml of 0.01 M potassium superoxide, 0.15 M dieyclohexyl.18-crown-6 (in DMSO) into 2.0 ml of 10 -~ M NBT.10-4 M EDTA in 0.05 M carbonate buffer pH 10.2 was measured in the absence and in the presence

of the bovine superoxide dismutase and metal complexes. We have employed Cu(II)-triglycine 31 1 : 1 complex for a control of the superoxide dismutation active substances. Plots of the absorbance versus the logarithm of the different concentrations of the Cu(II)-31 complex were linear. The concentration of the 31 complex required for a 50% inhibition of the reduction of NBT was determined graphically [30] to define 1 unit ( 0 . 4 . 1 0 - a M with the xanthine.xanthine oxidase assay and 0.2 • 10 -a M with the potassium superoxide assay). In the assay of a testing chelate, the absorbances were measured at five different concentrations, which were ascertained to give a linear plot of absorbance versus log concentration. The equivalent absorbance produced by 1 unit of the Cu(II)-31 complex was taken as 1 unit of the testing chelate. The measurements were repeated three times f o r each system and the mean values were taken. The deviation were within +-20% (Table I). In the two assays, all of the macrocyclic ligands alone showed no superoxide dismutase activity. Uncomplexing Cu(II) ions with macrocyclic ligand, which may bind to other potential ligands such as bovine serum albumin, superoxide dismutase etc, interfered with the potassium superoxide assays. To

176 nullify the inhibitory action of the uncomplexing Cu(II) or Ni(II) ions, ethylenediamine diacetate ligand (EDDA) was added as a sequestering agent. Separately we confirmed no superoxide dismutase activity with the Cu(II)- or Ni(II)-EDDA complex. Displacement of the macrocycles for EDDA is most unlikely, since the macrocyclic complexes are much more stable due to the 'macrocyclic effects' [14-16, 26,27]. It should be noted that a much stronger chelating agent EDTA cannot replace the macrocyclic polyamines except for the triamines such as 19 [31, 32]. We confirmed that the macrocyclic complexes by themselves did not interact with nitroblue tetrazolium in the adopted concentration range. Results and Discussion

Copper(II) complexes of low molecular weight amino acids (e.g. lysine, tyrosine, histidine) [17,19, 23], oligopeptides [17,18], and salicylates [20,22, 24] were previously shown to interact with superoxide, showing the activities in superoxide dismutase assays with the xanthine-xanthine oxidase system and potassium superoxide system. We confirmed the activity of some of these models and the bovine superoxide dismutase and determined their 1 unit values with the present assays. The activity shown by the xanthine-xanthine oxidase system is not the same as the one determined by the potassium superoxide system. However, they showed a similar activity trend with the macrocyclic polyamine complexes and, accordingly, we ascertained that the chelates are able to inhibit the reactions involving the superoxide anion. For the present study, we examined Cu(II)-gly, -glygly, -glyglygly (31), and -glyglyglygly (32) complexes. The latter two peptide complexes significantly showed a higher superoxide dismutase activity than the former two or previously known amino acid complexes, (see Table I). Only the doubly (with 31, for the complex CuH-2L structure, see Fig. 2a [33]) and triply deprotonated (with 32) peptide complexes have effective redox potentials, E, for Cu(II)~-Cu(III) (0.67 V and 0.38 V versus standard Calomel electrode, respectively) at physiological pH such that O2 oxidation to Cu(III) ts possible [34,35]. The easier access to Cu(III) state might be indirectly linked to the favorable O~ dismutation. Copper(II)

ion is the active center of galactose oxidase that also possesses the superoxide dismutase function [36]. It's site consists of four in-plane nitrogen atoms [36], or of two histidine imidazoles and two oxygen atoms in a pseudo-square-planar structure [37]. Its E value found was so low (0.41 V) that Cu(III) species was once considered to be involved in the enzymic reactions [38], although later this postulation was discarded [39]. The trivalent state of nickel can also be greatly stabilized by the similar square-planar coordination of the triglycine (31) and tetraglycine (32): their E values, respectively, are 0.85 V and 0.79 V versus standard Calomel electrode [40]. However, they did not show the superoxide dismutase activity in the present experiment. The triglycine complexes are structurally similar to our dioxotetraamine macrocyclic complexes, which have the two amide protons dissociated to enclose M(II) in square-planar geometries (Fig. 2b) [14,15]. The resulting physical and chemical properties are also alike: the most typically, the E values for Cu(II)and Ni(II).dioxotetraamine complexes are greatly lowered (e.g. 0.42 V versus standard Calomel electrode for Cu-1 and 0.62 V for Ni-1) and to stabilize MOII) states (the extent of the stability varies with the ring size) [16]. Therefore, the Cu(II)-triglycine 31 complex was chosen as a suitable reference in the superoxide dismutase activity test of various dioxopolyamine complexes. Among Cu(II) and Ni(II) complexes of macrocyclic polyamines and the relevant ligands 1-30 examined, the majority of the superoxide dismutase active species were dioxopolyamines; compare, for instance, 1 and 23, or 15 and 30. This may be rationalized by the fact that the deprotonated dioxopoly-

Cl

b

\o Cu2+-Trigly (31)

o

/...

ou2 M Cu2+- 1

Fig. 2. Schematic presentation of CuH-2L complex structures of (a) Cu(II)-31 and (b) Cu(II)-l.

177 amine ligands (containing two N atoms) can donate more electrons to M(II) than the dioxo-free counterparts (composed of only neutral N atoms) to stabilize M(III) states. The activities for the dioxopolyamine systems were in general comparable to those for the Cu(II)-oligopeptide (31 and 32) complexes in terms of the 50% inhibition of nitroblue tetrazolium reduction (i.e. 1 unit value). There are some deviations of the activities among the dioxopolyamines depending on the macrocyclic ring size (e.g. while 12-(1), 15(6) and 16-membered dioxotetraamine (9) systems are active, 13- (2) and 14-membered (3) homologues are not), donor atom (cf. 10, 15 and 16), substituent on the ring (of. 6, 7, and 8) or metal ion (e.g.while Cu(II)-I complex is active, Ni(II)-I complex is inactive). Interestingly, the activities are not strictly parallelling with the stability of Cu(III) (i.e. magnitude of E values). Cu(II)-2 and -3 doubly deprotonated complexes (CuH_2L) are extremely stable, since the planar N4 ring cavities of the 13-(2) and 14-membered macrocycle (3) fit to the Cu(II) cation size [14]. The magnitude of their stabilities log KCuH_2L (= [CuH-=L°] [L]) of -2.2 M-' (for 2) and 1.0 M-1 (for 3), and the location of their d-d absorption maxima, Xmax, of 520 nm (2) and 505 nm (3) indicate the analogies in the coordination geometry to the glycylglycylhistidine complex having log KCuH_2L of --2.1 and ~kmax of 525 nm [14]. Its redox potential E was reported to be 0.73 V versus standard Calomel electrode [34,35 ]. We found that the Cu(II)-glyglyhis complex, like Cu(II)-2 and -3, has no superoxide dismutase activity. This fact, along with the observation of the superoxide dismutase activities with. realtively unstable homologues Cu(II)-I and -4 [16], implies that too stable square-planar geometries may not be favorable for the Oi dismutation which may require some alteration of the coordination geometry. With copper(II) a pyridine-containing dioxopolyamine (15) and dimeric macricycles (17 and 18) capable of encapsuling two metal ions in the cavities (Kimamura, E., unpublished data) showed stronger activities. The n-electrons in the pyridine rang may favorably respond to the Cu(II) - O~ interaction. A molecular model of the complexes of 17 and 18 (see Fig. 3) suggests that the two encompassed Cu(II) ions can come close for the metal-metal interaction, which

might serve to promote the electron-transfer between Oi and the copper ions. With triamine ligands, dioxo-free macrocyclic 20

(Cu2+)2 - 18

Fig. 3. A proposed structure of Cu(II)2-18 complex.

and linear 21 and 22 showed the superoxide dismutase activity. An uncoordinated equatorial position in these complexes would be used for quenching O~ ion. Moreover, the relatively loose complex stuctures would work favorably for stereochemical changes accompanying the copper oxidation states. The superoxide dismutase inactivity of a 9-membered trairnine 19 complex may result from the very rigid and distored coordination [41] to render the remaining uncoordinated site electronically or sterically unreactive. It is appropriate to consider here a previous report [42] that coordinatively unsaturated Cu(II) complexes (such as diamines or triamine system) have catalase activity (i.e. they can interact with peroxide ion O]-), while coordinatively saturated tetraamine complexes (such as 25) have no catalase activity. The most interesting of all are Ni(II) complexes of 16-membered dioxopentaamines 11-14 (the structure is shown in Fig. 4) which are extraordinarily superoxide dismutase active. In a previous study (unpublished data), they exhibited extraordinarily low E values at approx. 0.24 V versus standard Calomel electrode for Ni(II) ~ Ni(III) under the slightly alkaline pH regions, which should be linked to the present observation of the extraordinary superoxide dismutase activity. Of further interest is the dramatic effect of substituents on the superoxide dismutase activity despite that they all show the same E values (see Table I). The substituent effect, therefore, may be steric origin rather than electronic one. The most

178 active naphthylmethyl substituted system (13)is more than 40 times as active as the reference Cu(II)triglycine (31) complex (by the xanthine-xanthine

ity correlations and also enhance the lipophilicity of the complexes for biological adaptation. More effort in the chemical modifications are underway in this laboratory to find compounds that are suitable enough to permit further therapeutic applications.

Acknowledgement This work was partially supported by a Grant-inAid for Scientifik Research from the Ministry of Ecucation, Japan (No. 447113).

Ni 2+- 11 Fig. 4. An established (Kimamura, E., unpublished data) structure of Ni(II)-ll complex. oxidase assay). Its activity, furthermore, surpasses that of Cu(II)-phenanthroline complex Cu(phen)~ ÷, which previously was shown to be a good quencher of O~ [29]. In summary, the present study has demonstrated that the complexes of macrocyclic dioxopolyamines certairdy possess superoxide dismutase activities and that appropriate modification of their chemical structures can improve the biological activities. Our low molecular weight compounds would merit further extensive investigations for biological applications for the following reasons. First, the macrocyclic eonplexes are extremely stable and won't readily decompose. They are hardly displaced with potential biological ligands such as proteins under physiological conditions. In this regard, our system is superior to oligopeptides such as 31 or 32 which tend to be exchanged by macro bioligands. Secondly, the macrocyclic complexes are in general soluble enough in aqueous solution and pose no solubility problem. Though many copper(II) chelates have the comparable superoxide dismutase activities (for salicylate, see Table I), a main problem with these known compounds (e.g. bis(salicylato)copper(II) [21,22] or 2.formypyridine thiosemicarbazone copper(II) [9-111 is their lack of solubility, which places a limit to their therapeutic applications [8]. Thirdly, the chemical modification of the basic macrocyclic structures is fairly easy, as exemplified by the introduction of phenylmethyl, naphthylmethyl and alkyl ~ubstituents on the 14(3--5), 15- (6--8) and 16-membered polyamines (1114). Thus, one could easily pursue the structureactiv-

References 1 McCord, J.M. and Fridovich, I. J. Biol. Chem. (1969) 244, 6049-6055 2 MeCord, M.M. Sciences, (1974) 185,529-531 3 Menander, K.B. and Huber, W. (1977) in Superoxide and Superoxide Dismutase (Michelson, A.M., McCord, J.M. and Fridovich, I., eds.), p. 537, Academic Press, London 4 Sorensen, J.R.J. (1976) J. Med. Chem. 19, 135-148 5 Dionisi, D., Galeotti, T., Terrannove, T. and Azzi, A. (1975) Biochim. Biophys. Acta 403,292-300 60berley, L.W., Bize, I.B., Sahu, S.K., Lenthauser, S.W.H.C. and Gruber, H.E. (1978) J. Natl. Cancer Inst. 61,375-378 7 Yamanaka, N.Y., Ota, K. and Utsumi, K. (1978) in Biochemical and Medical Aspects of Active oxygen (Hayaishi, O., ed.), pp. 183-190, University Park Press, Baltimore 80berley, L.W. and Buettner, G.R. (1979) Cancer Res. 39, 1141-1149 9 Antholine, W.E., Knight, J.M. and Petering, D.H. (1976) J. Med. Chem. 19, 339-341 10 Coats, E.A., Milstein, S.R., Holbein, A., McDonald, J., Read, R. and Petering, H.G. (1976) J. Med. Chem. 19, 131-135 11 French, F.A. and Blanz, E.J.J. (1974) J. Med. Chem. 17, 172-181 12 Kimura, E. and Yatsunami, T. (1980) Chem. Pharm. Bull. Jap. 28,994-997 13 Igarashi, K. Kashiwagi, K., Kakegawa, T., Hirose, S., Yatsunami, T. and Kimuri, E. (1980) Bioehim. Biophys. Acta 633,457-464 14 Kodama, M. and Kimura, E. (1979) J. Chem. Soc. Dalton Trans. 325-329 15 Kodama, M., Yatsunami, T. and Kimura, E. (1979) J. Chem. Soc. Dalton Trans. 1983-1788 16 Kodama, M. and Kimura, E. (1981) J. Chem. Soc. Dalton Trans. 694-700 17 Joester, K., Jung, G., Weber, U. and Weser, W. (1972) • FEBS Lett. 25, 25-28 18 Brigelius, R., Spottl, R., Bors, W., Lengfelder, E., Saran, M. and Weser, U. (1974) FEBS Lett. 47, 72-75

179 19 Younes, M. and Weser, U. (1976) FEBS Lett. 6 1 , 2 0 9 212 20 Younes, M., Lengfelder, E., Zienan, S. and Weser, U. (1978) Biochem. Biophys. Res. Commun. 81,576-580 21 De Alvare, L.R., Goda, K. and Kimura, T. (1976) Biochem. Biophys. Res. Cummun, 69, 687-694 22 Weser, U., Richter, C., Wendel, A. and Younes, M. (1978) Bioinorg. Chem. 8, 201-213 23 Weinstein, J. and Bielski, B.H.J. (1980) J. Am. Chem. Soc. 102, 4916-4919 24 O'Youn8, C. and Lippard, S.J. (1980) J. Am. Chem. Soc. 102, 4920-4924 25 Yatsunami, T., Sakonaka, A. and Kimura, E. (1981) Anal. Chem. 53,477-480 26 Kodama, M., Kimura, E. and Yamaguchi, S. (1980) J. Chem. Soc. Dalton Trans. 2536-5238 27 Kodama, M. and Kimura, E. (1978) J. Chem. Soc. Dalton Trans. 1081-1085 28 Misra, H.P. and Fridovich, I.J. (1972) J. Biol. Chem. 247, 188-192 29 Valentine, LS. and Curtis, K.B. (1975) J. Am. Chem. Soc. 97,224-226 30 Imanari, T., Hirota, M., Miyazaki, M., Hayakawa, K. and Tamura, Z. (1977) Igaku no Ayumi, (in Japanese) 101, 496-497 31 Kodama, M. and Kimura, E. (1978) J. Chem. Soc. Dalton Trans. 248-253 32 Kodama, M., Yatsunami, T. and Kimura, E., (1980)

Inorg. Chem. 19, 1600-1602 33 Freeman, H.C. (1967) in Advances in Protein Chemistry (Anfinsen, C.B., Anson, M.L., Edsall, J.T. and Richards, F.M., eds.), Vol 22, p. 257, Academic Press, New York 34 Bossu, F.P., Challapa, K.L. and Margerum, D.W. (1977) J. Am. Chem. Soc. 99, 2195-2203 35 Margerum, D.W., Wong, L.F., Bossu, F.P., CheUappa, K.L., Czarnecki, J.J., Kirksey, S.T., Jr., and Neubecker, T.A. (1977) Bioinorgaric Chemistry, Advances in Chemical Series 162, p. 281 Vol. II Am. Chem. Soc. 36 Cleveland L. and Davis, L. (1974) Biochim. Biophys. Acta 341,517-523 37 Bereman, R.D. and Kosman, D.J. (1977) J. Am. Chem. Soc. 99, 7322-7325 38 Hamilton, G.A., Adolf, P.K., De Jersey, J., Debois, G.C., Dyrkacz, G.R. and Libby, R.D. (1978) J. Am. Chem. Soc. 100, 1899-1912 39 Winlder, M.E. and Bereman, R.D. (1980) J. Am. Chem. Soc. 102, 6244-6247 40 Bossu, F.P. and Margerum, D.W. (1976) J. Am. Chem. Soc. 98, 4003-4004. Inorg. Chem., 16 (1977) 12101214 41 Bereman, R.D., Churchill, M.R, Schaber, P.M. and Winlder, M.E. (1979) Inorg. Chem. 19, 3122-3125 42 Sigel, H. (1969) Angew. Chem. Int. Edn. 9, 167-177 43 Palmer, B.N. and Powell, H.K.J. (1974) J. Chem. Soc. Dalton. Trans. 2089-2092