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Active site-specific reconstituted copper(II) horse liver alcohol dehydrogenase: A biological model for type 1 Cu2+ and its changes upon ligand binding and conformational transitions
Active Site-Specifk Reconstitutbd Copper Horse Liver Alcohol Dehydrogenase: A Biollogigical Model for Type 1 Cu*+ and Its Changes Upon Ligand Binding and Confnrmational Transitions Wolfgaug Maret, ’ Hehuut Dietrich, ’ Hans-HeiurichRuf,* aud Michael Zeppezauer’ IFachbereich 15.2. AnaljtiMe md Biologische Chemie. Universitiir &s Saarkutaks, 06600 Saar&iicken Il. and 2Fachbereich 3.13. Physiologische Chemie, Universiriir ties SaarZan&s, D-6650 ffombur&zar, West Germany ABSTRACT Insertion of Gun+ ions into horse liver alcohol dehydrogenase depleted of its catalytic Znz* ions creates an artifkiai blue copper center sin&r to that of plastocyanin and similar copper proteins The en spectrum of a frozen solution and the optical spectra at 296 and 77 IC am reported, together with the corresponding data for binary and ternary complexes with NAD+ and pyrazole. The binary complex of the cupric enzyme with pYrazole estabhshes a novel type of copper proteins having the optical characteristics of Type 1 and the esr parameters of Type 2 Ct@+. Ternary complex formation with NAD+ converts the Ct@ ion to a Type 1 center. By an intramolecuIar redox reaction the cuprous enzyme is formed from the cupric enzyme Whereas the activity of the cupric alcohol dehydrogenase is difficult to assess (0.5%-l% that of the native enzyme), the cuprous enzyme is distinctly active (8% of the native enzyme). The implications of these fmdhrgs are discussed in view of the coordination of the metal in native copper proteins.
INTRODUCTION The four zinc ions in horse liver alcohol dehydrogenase (EC 1.1.1.1) are bound in two kinds of binding sites with different coordination environments and functional properties_ The catalytic zinc ion is coordinated in a distorted tetrahedral geometry by two sulfur, one nitrogen and one oxygen atom (Cys 46, Cys 174, His 67, and water); the noncatalytic zinc ion is coordinated by four sulfur atoms (Cys 97,100,103, and 111) JownalofInoganicB&hemirtry 12,241-252 6 JZlsevierNorth Hohand, Inc., 1980
(1980)
52 Vanderbilt Ave., New York, New York 10017
241 0162-0134/80/030241-12gO2.25
Wolfgang Maretet aL
242
[I] _ Each of these binding sites resembles the metal binding locus of an electron transport protein. The noncatalytic site is similar to the iron binding site in rubredoxin [2] _ The catalytic site resembles that of Type 1 copper proteins such as plastocyanin [3] _ Insertion of Cu2* ions in the cataIytic site of horse liver alcohol dehydrogenase may serve the dual purpose of introducing a spectroscopic probe for studying the catalytic mechankn
of the enzyme and providing a model system for copper proteins with a
structurally defmed environment. This paper reports the preparation and spectral properties of horse liver alcohol dehydrogenase with Cu2* in the catalytic site. MATERIALS
AND METHODS
Crystalline horse liver alcohol dehydrogenase (EC 1.1.1 .l), NAD+, and NADH were purchased from Boehringer-Mannheim. All chemicals were of analytical reagent or spectroscopic grade. Metal analyses were carried out by atomic absorption spectroscopy using i Perkin-Elmer 400 atomic absorption spectrophotometer. The enzyme depleted of catalytic zinc ions (H~Zn(n)2-enzyme) was prepared as described previously [4]. Cupric ions were inserted into this crystalline enzyme by stoichiometric addition of cupric sulfate or bis~ethylenediarnine)copper(II) in 0.025 M Tes/Na+, pH 7.0. Tbereafter, the copper alcohol dehydrogenase crystals were dialyzed exhaustively against the crystallization buffer. The preparation was done at 277 K under nitrogen atrnosphere. Activity was determined according to Dalziel [S] _ For each preparation the activity was compared to that of the H&?!n(n)+mzyme which had not been subjected to insertion of cupric ions. The optical spectra were recorded with a Hitachi-PerkinElmer 556 spectrophotometer at 296 K. The esr spectra were measured on a Varian E-9 X band spectrometer at 100 K_ The modulation frequency was 100 kHZ, the microwave power was 20 mW, and the modulation amplitude was 5 G. The esr parameters were estimated by direct measurements from the recorder tracings_ NomencIatur@ The catalytic metal is denoted by “8
and the noncatalytic metal by “rz” after the
chemical symbol of the metal, i.e., H4Zn(n)2-enzyme: enzyme depleted of catalytic metal ions_ Co(c),Zn(n),-errzyme: enzyme whose catalytic zinc ions are replaced by Co2+. Cu(c)~Zn(n&mzyme:
enzym e whose catalytic zinc ions are.replaced by Cu2+.
RESULTS
chemical and Spectral Properties of Cu(c), _e Zn(n), _a-Horse Liir
Alcohol Dehydrogenase
The reconstitution of the catalytic metal binding site of horse liver alcohol dehydrogenase with cupric ions turned out to be a slow process. After at least two days of incubation of a suspension of crystals of the H,Zn(n),_aenzyme with a stoichiometric amount of bis-(ethylenediamine)copper(iI), the crystals had become deep blue. According to the metal analyses, 0.6-13 g-atom copper had been incorporated per mole 1 Abbreviations: Tes, N-Tris-(hydroxymethyl)methyi-2-amiuomethane charge tmnsfer.
sulfonic acid LMCT. Jigand-to-metal
Copper(D) Horse Liver Alcohol Dehydrogenase
243
of enzyme. The degree of reconstitution was lower when cupric sulfate was used instead of bis-(ethylenediamine)copper(II)_ The spectral properties were identical in both cases. The esr and optical spectra were measured with a preparation of the composition Cu(c)o_eZn(n)l_aenzyme_ The ultraviolet spectrum was checked against the protein concentration as determined by the method of Lowry [6], using the zinc enzyme as standard. The same extinction coefficient was found for the blue and for the native enzyme as long as the cupric enzyme retained its blue color. The activity of the cupric enzyme was OS%-1% that of the native zinc enzyme. In this vahre, the lower occupancy of the catalytic metal binding sites in the cupric enzyme is not taken into account. Since the H4Zn(n)senzyme shows residual activity between 0% and OS%, it cannot be decided definitely whether the cupric enzyme is to be regarded as inactive or slightly active. Whereas the crystals retained their color on standing under nitrogen, the solution of the cupric enzyme turned almost colorless under a period of 24 h. The activity of this colorless enzyme increased to about eight percent. (This would amount to about 20% in a fully substituted cuprous alcohol dehydrogenase). The colorless enzyme could be reconverted to a blue species upon addition of potassium hexacyanoferrate(II1). We interpret the change of color and activity of the cupric enzyme as being due to an intramolecular redox reaction yielding the cuprous enzyme. Possibly, a disulfide bridge is formed between cysteines 46 and 174. This would enable the cuprous ion to remain bound in the catalytic site. CompIexes of Cul* with disuhides are well known [7] _ However, the stoichiometry of this reaction remains unclear, since the reduction of the cupric ion requires one electron, whereas the formation of the disulfide bridge yields two electrons. As illustrated in figure I, the conversion of the cupric into the cuprous ion is accompanied by the occurrence of LMCT transitions in the ultraviolet spectrum of the enzyme below 300 nm similar to those observed in copper metallothioneine [S] _The optical
and esr spectra
of the cupric enzyme
are shown in figures 2 and 3 (upper trace)_
In Tables I and 2 the parameters of optic& and esr spectra are given, together with relevant data from naturally occurring blue copper proteins. These data show convincingly, that the state of the copper in the blue Cu(c)o_eZn(n)1_8-enzyme can be described as similar to Type 1 Cu 2+ of the blue copper proteins [9] _The visible spectrum of the blue Cu(c) o.eZn(n)l.aenzyme is similar to that of azurin (see Table 1) [lo], while the esr spectra indicate that the cupric enzyme appears to be similar to stehacyanin [9] , rusticyanin [ 111 and plantacyanin [12] due to the occurrence of rhombic components in the esr spectrum (see figure 3, upper trace)_ These components are almost absent in plastocyanin, azurin, and umecyanin [9, 131. Furthermore in the cupric enzyme Iigand hyperfine structure (most probably due to His 67) is observed in the esr spectrum (see figure 3, upper trace)_ When measuring the esr spectra of the unhganded cupric enzyme it was observed that upon cooling to 77 K the bIue color changed to violet. The optical spectrum of the cupric enzyme at 77 K is shown in figure 4 (solid line). The low-energy band is better resoIved at 77 K; the main band is blue-shifted 40 nm upon cooling. The blue color reappears again on warming to 296 K. A detailed analysis of this temperature dependence has not yet been made.
Wolfgang Maret et aL
244
FIGURE L Ultraviolet eIectronic absorption spectra of cupric (right) and cuprous (left) horse liver alcohol dekydrogenase in 25 mM TesjNa+, pE 69_ Concentration of enzyme: 8.3 & (right) and 26 pM (left).
Effect of Coenzyme and Inhiiitor on the Spectra of Cu(c)o_eZn(n)I_a-Horse Alcohol ikhydmgenase Lie Binary Complexes with NAP
Liver
or &razo!e
a Oxidked coenzymk Binding of NAD+ causes a large redshift of the charge transfer band from 620 to 650 mn at 296 K and from 580 to 595 mn at 77 K (Figure 5, solid line and Figure 7, solid line). This resembles the behavior of the Co(c)aZn(n)senzyme [14] - Since the coenzyme is not coordinated directly to the catalytic metal ion [2], a conformation change is likely to occur which affects the geometry of the catalytic metal binding site. Also, the esr spectrum (Figure 6, upper trace) indicates some change occurring in the metal binding site. The superhyperfme interaction of nitrogen is much less resolved upon binding of the coenzyme. However, the data in figures $6, and 7 and Tables I and 2 showthat even in the binary complex with NAD+ the state of the copper can still be described as Type 1 Cu*+. b. Pymzoie: This ligand acts as a competitive inhibitor of ethanol oxidation and coordinates directly to the catalytic zinc ion in the native enzyme [ 15]_ In the absence of coenzyme, the esrspectrum becomes axial with concomitant increase of Ai from 30 to 115 X 10m4 cm-l. This indicates a change of the state of the copper from its Type I FIGURE 2. Visiile electronic absorption spectrum of cupric horse liver alcohol dehydrogenasein 25 mM Tes/Na+, pH 6.9 at 296 K. Concentration of enzyme: 630 pIK
Copper(D)
Horse Liver Alcohol Dehydrogenase
245
.___‘._A_‘_.
2600
k__
ax!0
.
_.
3.00
G
FIGURE 3. esr spectra of native cupric horse liver alcohol dehydrogenase (upper trace) and of its binary complex with pyrazole (lower trace) recorded at X-band frequency. Temperature: PO0 K; concentration of enayrne: 630 &f in 25 rnM Tes/Na*, pH 6.9; concentration of pyrazole: 40 mh4.
upon binding of pyrazole. Ligand byperfime structure (AIN = 14 X 10-a cm-l) due to two nonequivalent nitrogen nuclei (i.e., the imidazole of His 67 and of pyrazole) is observed in the go region of the esr spectrum (Figure 3, lower trace). Upon binding of pyrazole to the enzyme a marked acceleration of the reduction of the cupric ion is observed at 296 K. Therefore the intensity of the bands in the visible spectrum of the binary complex could not be measured accurately. No shift of the 620 run band was observed at 296 K. On cooling to 77 K;the color of the enzyme changes from blue to pink (Figure 4, broken line). When the binary com- plex was cooled immediately after mixing of the cupric enzyme with pyrazole, the blue color reappeared on warming to 296 K. cJtaracter&ics
TABLE
1: Absorption Maxima of Cupric Horse Liver Alcohol Dehydrogenase and its Complexes in Comparison with Azurin and StellacyanirP A,,,&m)
&lYU (nm) at 296 Kc 1 native enzyme 2 binary complex with NAB+ 3 binary complex with pyrazole 4 ternary complex with pyrazole and NAD+ s azurin 6 stellacyanin
377 442 (E = 600) (E = 500)
620 (E = 2000)6
at 77 Kc
365 430 580 755
435
650
365 420 595
387
432
620
405 495 690
390
460
550
690
563
627
385 460 535 680
480
(E = 185) (E = 504)
443 560 (E = 942) (E = 1542)
(e = 3798) 603 (E = 3549)
u Taken from Ref. 10. b Extrapolated to one g-atom Cu*+ per subunit on the basis of both the experimentally determined metal content and the protein coneentratioa c Values of e givenin (M-1 cm-l).
Wolfgang Maret et aL
246
TABLE 2t Spin-Hamilt~nianParametersof Cupric Horse Liver Alcohol Dehydrogenaseand its Complexes. For Comparison the Data of Azurin, Stellacya.ni+ and a Low Molecular Weight Compoundb are also Listed
1 native cupricenzyme 2 binary complex with NAD+ 3 ternary complex with NAD+ and pyrazok ‘4 steuacyanin 5 z3.zoG 6 binary complex with 7
32 36 56
pyrazole
115
bM35dimethylpyxazole-lcarbodithioato)coppe~~
160
Data taken from Ret 9b Data taken from Ref. 24. C Values of A givenin ( lW4
14
30 32
56 7
15
224 2.29 226
208 206
14
2.20
205
15
216
29 7
15
2.25 2-24
2.03
2.04
2.07
0
cm-l).
The Ternary CompItx with NAD’ antipymZoIe. The native zinc enzyme forms strong ternary complexes with reduced coenzyme and fatty acid amides or with oxidized coenzyme and pyrazole [1] _ In the enzyme reconstituted with cobalt in the catalytic site the same binary and ternary complexes are also formed j14]. In the cupric enzyme we studied only the ternary complex formed upon binding of NAIY to the binary enzyme-pyrazole complex. The esr measurements (Figure 6, lower trace) indicate that the state of the copper changes again from the novel type to Type 1 Cu2+. The ternary complex is blue at 296 and 77 K (Figure 5, dotted line and Figure 7, broken line)_ Thus, ternary complex formation seems to stabilize the Type 1 state of the catalytic cupric ion within a wide range of temperature_
FIGURE 4. Viiile electronicabsorptionspectraof cuprichorseliveralcohol dehydrogenase(solid line) and its binary compkx with pyrazole (broken line) in 25 mM T-a+, pH 6.9 at 77 K; concentrationof enzyme: 630 /.M; concentrationof pyrazole: 40 mM.
CoPPerfII) Horse Liver Alcohol
Dehydrogenase
az-
u-
I UIO
I 500
I 600
I 700
I 800
hhnl FIGURE 5. Visible electronic absorption spectra of cupric horse liver alcohol dehydrogenase in its binary complex with NAD+ (solid line) and ternary complex with NAD+ and pyrazole (dotted line) in 25 mM Tes/Na*, pH 6.9 at 296 K; concentration of enzyme: 109 uM; concentration of NAW: 632 mM; concentration of pyrazoler 9.9 mM.
8
‘
2600
3000
I
3Lal
1
G
FIGURE 6. esr spectra of cupric horse liver alcohol dehydrogenase in its binary complex with
. . NAD+ (upper trace) and ternary complex with NAD* and pyrazole (lower trace) recorded at X-band frequency; temperature: 100 K; concentration of enzyme: 630 /.&f in 25 mM Tes/Na+, pH 6.9; concentration of NAD+: 2.3 n&f; concentration of pyrazole: 3.8 mM.
Wolfga_ng Maret et aL
248
FIGURE 7. Visiile electronic absorption spectra of cupric horse liver alcohol dehydrogenase in its binary complex with NAD+ (solid line) and temary complex with NAD* and pyrazole(broken line) in 2.5 mM T.es/Na*, pH 6.9 at 77 K; concentration of enzyme: 630 /.&f; concentration of NAD*r 2.3 rri!!; concentration of pyrazole: 3.8 mM.
Coxnparisonof the Binding Sites of the Blue Copper Proteins and Alcohol Deplydrogenase An important difference between the genuine Type 1 copper proteins and the cupric enzyme is the occurrence of mo cysteine residues as metal ligands in the latter. This probably is the reason for the slow conversion into a cuprous enzyme. In the genuine Type 1 copper proteins nature has elegantly circumvented the possible formation of a dimbide bridge within the metal binding site by choosing cysteine and methionine as metal ligands together with two histidine residues, as has been found in plastocyanin and azurin [3,16] _ The conserved tetrahedral binding site upon removal of the catalytic zinc ion would not only provide the proper environment for the Cua+ ion to render a Type 1 Cu2+ center but also would offer the most favorable site for Cul+ ions that prefer tetrahedral complexes. In this context, the enhanced enzymatic activity of the cuprous alcohol dehydrogenase is remarkable_ This was observed after an internal oxidation-reduction process which yielded cul+ in the catalytic site and possibly a disulfide bridge between Cys46 andCys 174. Cleariy, the preparation and examination of a cuprous enzyme built up from Cul+ and the H4Zn(n)2enzyme is highly desirable.
Binding of PyrazoIe Creates a Novei Type of Cu2+ Protein As in the native horse liver alcohol dehydrogenase and the cobalt substituted enzyme the cupric Cu(c),-,GZn(n)I_8enzyrne binds pyrazoIe by coordination to the catalytic metal ion. In the native Type 1 Cu2’ proteins no binding of inner sphere ligands has been observed and the artificial cupric enzyme seems to be unique iu this respect_ The binding of pyrazole to form the binary complex changes the state of the cupric ion:
Copper(D) Horse Liver Alcohol Dehydrogenase
249
The data in Tables I and 2 demonstrate that the optical and esr properties of both the free cupric enzyme and of its ternary complex are similar to those observed for Type 1 Cu2+ proteins. In the binary complex with pyrazole, the optical and esr properties are not in accordance with any of the established states of Cu2+ in copper proteins_ Sakaguchi and Addison point out in a recent article [17] that the quotient g”/Al may be used as “a convenient empirical index of distortion of the donor set from planar toward tetrahedral.” In the unliganded cupric horse liver alcohol dehydrogenase the value gn/Alt = 750 cm is in accordance with those found for the Type 1 copper proteins_ In contrast, a value gH/Aa = 147 cm is found for the binary complex of the cupric enzyme with pyrazole. This ratio is characteristic of Type 2 Cu2+. However, the optical spectrum exhibits the intense charge-transfer characteristically found only in Type 1 Cu2* proteins. Also, the binary complex with pyrazole does not bind fluoride ions which has been regarded as a criterion of Type 2 Cuz+ sites [ 18 ] . It should be emphasized that the pyrazole complex of the cupric enzyme is chemically more similar to plastocyanin and azurin than the unliganded cupric enzyme, since the copper is bound to two sulfur and two nitrogen atoms. The different state of the cupric ion in the binary complex might therefore be ascribed to differences in the coordination geometry only, if we assume that pyrazole substitutes the metal bound water molecule. A nearly square planar arrangement seems reasonable for this species (cf. the gll/All values for the binary complex and the model 7).
The Catalytic Copper Ion, Although inactive, is a SensitiveReporter of Events Related to the Catalytic Cycle From numerous structural and kinetic studies on the native zinc alcohol dehydrogenase it is well known that binding of the coenzyme induces a major conformational transition in the protein prior to the binding of the substrate [l] _As a consequence of this transition, the catalytic metal ion becomes able to bind and to activate the substrate. Additional support for this sequence of events has been obtained from spectroscopic studies using active site-specific reconstituted cobalt alcohol dehydrogenase [ 19]_ Therefore it is promising to observe that the binding of NAD+ (and of NADH, unpublished results) to the copper enzyme alone gives rise to changes in both the optical and esr spectra which must be interpreted as distortions of the coordination sphere. A different set of spectra is obtained in the ternary complex with NAD+ and pyrazole. In both complexes, the state of the copper can be described as Type 1 Cu2+_ This means that in the framework of this coordination type the conformation changes induced by the coenzyme and subsequent formation of ternary complex with inhibitor are accompanied by certain changes in the coordination sphere of the catalytic cupric ion in terms of bond angles and probably also bond distances. In view of the negligible activity shown by the cupric enzyme it may be suspected that the coenzyme induced transitions in the protein structure are rather independent of the kind of metal present in the catalytic site. The metal seems to be generally involved in this conformation change, irrespective of its ability to activate the substrate. Evolutionary Aspects The similarity between the catalytic site in horse liver alcohol dehydrogenase substituted with Cu2+ ions and the copper binding site of certain blue copper proteins
WoIfgang Maret et al.
250
leads to the question of whether the catalytic zinc binding site of alcohol dehydrogenase and the copper binding sites of the blue copper proteins may share some common ancestors_ Common structural features observed in both classes of proteins are the occurrence of suIfur as metal ligapd and as far as available data reveal a distorted tetrahedral coordination geometry [3, 163 _The metal atoms in both plastocyanin and horse liver alcohol dehydrogenase are accessiile from two directions; one access is provided through a hydrophobic channel, the other one from a more hydrophilic environment, that is, the solvent in the case of plastocyanin and the anion binding site in the coenzyme binding domain of alcohol dehydrogenase. On the other hand, an important difference is obvious: The copper ion in plastocyanin is coordinated to four protein side- chains (Cys, Met, 2 His [3]) whereas alcohol dehydrogenase has three ligands from the protein (2 Cys, His [I]) and an exchangeable water molecule as fourth ligand: The participation of the metal in catalysis as it has been inferred from numerous studies seems to proceed via outer sphere interactions in the redox processes catalyzed by plastocyanin [3] and via direct coordination of the substrate to the catalytic metal ion in alcohol dehydrogenase [I91 _ Also, comparison of the tertiary structure of plastocyanin with the catalytic domain in horse liver alcohol dehydrogenase shows no obvious similarity_ This is in contrast to the folding of the polypeptide chain around the noncatalytic metal ion in horse liver alcohol dehydrogenase, which is similar to ferredoxin [2] _ It has already been pointed out that the catalytic metal binding sites of several zinc-containing enzymes including horse liver alcohol dehydrogenase show common features with respect to ligand composition and coordination geometry [20] _ Most probably, this is due to very general chemical principles governing the design of these zinc-binding sites rather than to evolutionary relationships_ In the particular case of alcohol dehydrogenase we believe that the occurrence of cysteine as ligand of the catalytic zinc in alcohol dehydrogenase, which seems to be a common property in this class of enzymes [21], is also dictated by the chemical reasons inherent in the catalytic mechanism and not by evolutionary relationships to blue copper proteins_ CONCLUSIONS The artificial copper protein Cu(c)zZn(n)z-horse liver alcohol dehydrogenase is a versatile biological model system for studying the unusual spectroscopic properties of native copper proteins. Moreover it is a flexible coordinative system that changes its coordination sphere upon interaction with ligands or due to conformational changes within the protein triggered by coenzymes. The redox behavior of copper(H) liganded to thiolate sulfur is another aspect to be studied with this cupric enzyme. The cupric liver alcohol dehydrogenase liganded with pyrazole has to be considered as a novel, probably planar type combining the esr characteristics of Type 2 and the intense absorption spectra of Type 1, although blueshifted. Thus square planar coordination wi’J1 sulfur ligands does not necessarily give rise to Type 1 behavior in terms of esr parameters and to Type 2 in terms of optical spectra. From our data pentacoordination, which has been proposed for the blue copper proteins [22] (and which is the most adequate description for Type 2 Cu2* [23]), for either the native cupric enzyme or its complexes cannot be exckded. The conserved tetrahedral geometry in the cupric enzyme and its complexes with coenzyme as compared to the native zinc enzyme demonstrates the usefulness of Cua+ as a reporter group. The Cu2+ is affected by the coenzyme triggered conformation
Copper(H)
Horse Liver Alcohol Dehydrogenase
251
change of the protein, which makes it possible to study details of the catalytic cycle even in the copper substituted species. Financialsupporr was obtained from Deutsche Forschungsgemeinschaft(Ze 1.5216) and For& der
Chemirchen liuiustrie. We appreciate discussions withProfessorsI. Bertini, W. Haase, H. Korbwsk< T. D. Luckey, M. M&&n,
G. Rot&,
and R. J. P. Willtims
REFERENCES 1. C.-I. Br%rd&, H. Jiimvall, H. Eklund, and B. Furugren, in The Enzymes. 3rd ed,. P. D. Boyer, Ed., Academic Press, New York and London, 1975, VoL XI, Chap. 3, p_ 103-190. 2. H.Ekhmd, B. Nordstrcm, E. Zeppezauer, G. Siiderlund, I. Ohlsson, T. Boiwe, B.-O. Soderberg, 0. Tapia, C-1. Bed&, and A. Akeson, Three-dimensional Structure of Horse Liver Alcohol Dehydrogenasc at 2.4 8, Resolution,L Mol. Biol. 102,27-59 (1976). 3. P. M. Coleman, H_ C. Freeman, J_ M. Guss, M. Murata, V. A. Norris, J. A. M. Ramshaw, and M. P. Venkatappa, X-ray Crystal Structure Analysis of Plastocyanin at 2.7 A Resolution, Nature 272,319-324 (1978). 4. W. Maret, I. Andersson, H. Dietrich, H. Schneider-BernRihr. R. Einarsson, and M. Zeppezauer. Site-Specific Substituted Cobalt(H) Horse Liver Alcohol Dehydrogenases, Eur. J. Biochem. 98,501-512 (1979). 5. K. Dalziel, The Assay and Specific Activity of Crystalline Alcohol Dehydrogenase of Horse Liver,Acta Chem. &and. 11,397-398 (1957). 6. 0. H. Lowry, N. 3. Rosenbrough, A. L. Farr, and R. J. Randall, Protein Measurement with the Folin Reagent,J. BioL c7rem. 193,265275 (1951). 7_ R &terberg. Models for Copper-Protein Interaction based on Solution and Crystal Structure Studies, Coord. Uzem. Rev. 12,309-347 (1974). 8_ H- Rupp and U. Weser, Conversion of Metallothionein into Cu-Thionein, the possible low molecular weight form of neonatal Hepatic Mitochondrocuprein, FEBS Lett. 44, 293-297 (1974). 9. A. S_ Brill. nansition Metals in Biochemisrry. Springer Veriag. Berlin and New York, 1977. 10. E. I. Solomon, J. W. Hare, and H. B. Gray, Spectroscopic Studies and a Structural Model for Blue Copper Centers in Proteins, Proc NatL Acad. Sci USA 73,1389-1393 (1976). 11. J. C. COX, R. Aasa and B. G. MahstrGm, EPR Studies on the Blue Copper Protein Rusticyanin, FEBSLett. 93,157-160 (1978). 12_ V- T. Aikazyarr and R. M. Nalbandyan, Piantacyanin from Spinach, FEBS Lett. S&272-274 (1975). 13. T. Stigbrand, B. G. Mahnstr~m, and T. V&nqard, On the State of Copper in the Blue Protein Umecyanin,FEBS Lett. 12,260-262 (1971). 14. H. Dietrich. W. Maret, 1. Andersson, and M. Zeppeaauer, Site-Specifically Substituted Cobalt Horse Liver Alcohol Dehydrogenase, Hoppe Seyler’s Z. Physiol. Chem. 359,1074 (1978). 15. H. Eklund. personal communication. 16. E. T. Edman, R. E. Stenkamp, L. L. Sicker, and L. H. Jensen, A Crystallographic Model for Azurin at 3 A Resolution,J. MoL B&L 123,3547 (1978). 17. U. Sakaguchiand A. W. Addison. Spectroscopicand Redox Studies of Some Copper(H) Complexes with Biomimetic Donor Atoms: Implications for Protein Copper Centers, J. cherry. SosocDa&on 1979,600-608. Cnemistry, R. J. P. Williams and J. R. R. F. 18. B. G. Malmstriim, in New ‘12ends in Bihnoaanic Da Silva, Eds., Academic Press, London and New York, 1978,Chap. 3. 19. H. Dietrich, W. Maret, L. Wall&, and M. Zeppezauer, Active SiteSpecific Reconstituted Cobalt@Il Horse Liver Alcohol Dehydrogenase: Changes of the Spectra of the Substrate Trans4N,l+Dlmethylaminocinnam aldehyde ar@ of the Catalytic Cobalt Ion Upon Ternary Complex Formation with NADH and 1,4,5,6-Tetrahydronicotinamide Adenirre Dinucleotide, Eur. L B-em., 10,267-270 (1979).
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20. P. Argos, R_ M- Gravito. W- Eventoff, bi. G. Rossmann.and C-1. BrZnde’n,SimilaritiesIn Active Site Center Geometries of Zinc-containingEnzymes, Proteasesand Dehydrogenases,1. MOLBioL 126.141-158 (1978). 21. H_ JamvaU, Differences between Alcohol Dehydrogenases,Eur. J. Biocitem 72. 443-452 (1971). 22 B. Mondovi, L. Morpurgo, G. Rotilio and k Finazzi-A&, in Iron and Copper J+ot&s, K. T. Yasunobu, H. F. Mower, and 0. Hayaishi,JZds.,A&. Exp. Med. BioL 74,424-437 (1976). 23. G. RotiIio, L. Morpurgo, L. Calabrese, A. Finazi-Agr& and B. Mondovi, in Metal-Lignnd Interactiims in Organic Chemimy rmd Biochemisrry. B. Pulbrmnand N. GoIdbIum Eds., D. Reidel PublishingCompany, DordrechtHolland, part I, p. 243-253 (1977). 24. R. D. Bereman,G. D. Shiekls,and D_ NaIewajek,Characterizationof the CoordinationProperties of Bis(3,S-dimethyIpyrazoI~l~bodithioato) copper(ID, Inog. C7zem 17,3713-3714 (1978).
ReceivedJwze II. 1979; revisedOctober 16.1979.
’
INTRODUCTION The four zinc ions in horse liver alcohol dehydrogenase (EC 1.1.1.1) are bound in two kinds of binding sites with different coordination environments and functional properties_ The catalytic zinc ion is coordinated in a distorted tetrahedral geometry by two sulfur, one nitrogen and one oxygen atom (Cys 46, Cys 174, His 67, and water); the noncatalytic zinc ion is coordinated by four sulfur atoms (Cys 97,100,103, and 111) JownalofInoganicB&hemirtry 12,241-252 6 JZlsevierNorth Hohand, Inc., 1980
(1980)
52 Vanderbilt Ave., New York, New York 10017
241 0162-0134/80/030241-12gO2.25
Wolfgang Maretet aL
242
[I] _ Each of these binding sites resembles the metal binding locus of an electron transport protein. The noncatalytic site is similar to the iron binding site in rubredoxin [2] _ The catalytic site resembles that of Type 1 copper proteins such as plastocyanin [3] _ Insertion of Cu2* ions in the cataIytic site of horse liver alcohol dehydrogenase may serve the dual purpose of introducing a spectroscopic probe for studying the catalytic mechankn
of the enzyme and providing a model system for copper proteins with a
structurally defmed environment. This paper reports the preparation and spectral properties of horse liver alcohol dehydrogenase with Cu2* in the catalytic site. MATERIALS
AND METHODS
Crystalline horse liver alcohol dehydrogenase (EC 1.1.1 .l), NAD+, and NADH were purchased from Boehringer-Mannheim. All chemicals were of analytical reagent or spectroscopic grade. Metal analyses were carried out by atomic absorption spectroscopy using i Perkin-Elmer 400 atomic absorption spectrophotometer. The enzyme depleted of catalytic zinc ions (H~Zn(n)2-enzyme) was prepared as described previously [4]. Cupric ions were inserted into this crystalline enzyme by stoichiometric addition of cupric sulfate or bis~ethylenediarnine)copper(II) in 0.025 M Tes/Na+, pH 7.0. Tbereafter, the copper alcohol dehydrogenase crystals were dialyzed exhaustively against the crystallization buffer. The preparation was done at 277 K under nitrogen atrnosphere. Activity was determined according to Dalziel [S] _ For each preparation the activity was compared to that of the H&?!n(n)+mzyme which had not been subjected to insertion of cupric ions. The optical spectra were recorded with a Hitachi-PerkinElmer 556 spectrophotometer at 296 K. The esr spectra were measured on a Varian E-9 X band spectrometer at 100 K_ The modulation frequency was 100 kHZ, the microwave power was 20 mW, and the modulation amplitude was 5 G. The esr parameters were estimated by direct measurements from the recorder tracings_ NomencIatur@ The catalytic metal is denoted by “8
and the noncatalytic metal by “rz” after the
chemical symbol of the metal, i.e., H4Zn(n)2-enzyme: enzyme depleted of catalytic metal ions_ Co(c),Zn(n),-errzyme: enzyme whose catalytic zinc ions are replaced by Co2+. Cu(c)~Zn(n&mzyme:
enzym e whose catalytic zinc ions are.replaced by Cu2+.
RESULTS
chemical and Spectral Properties of Cu(c), _e Zn(n), _a-Horse Liir
Alcohol Dehydrogenase
The reconstitution of the catalytic metal binding site of horse liver alcohol dehydrogenase with cupric ions turned out to be a slow process. After at least two days of incubation of a suspension of crystals of the H,Zn(n),_aenzyme with a stoichiometric amount of bis-(ethylenediamine)copper(iI), the crystals had become deep blue. According to the metal analyses, 0.6-13 g-atom copper had been incorporated per mole 1 Abbreviations: Tes, N-Tris-(hydroxymethyl)methyi-2-amiuomethane charge tmnsfer.
sulfonic acid LMCT. Jigand-to-metal
Copper(D) Horse Liver Alcohol Dehydrogenase
243
of enzyme. The degree of reconstitution was lower when cupric sulfate was used instead of bis-(ethylenediamine)copper(II)_ The spectral properties were identical in both cases. The esr and optical spectra were measured with a preparation of the composition Cu(c)o_eZn(n)l_aenzyme_ The ultraviolet spectrum was checked against the protein concentration as determined by the method of Lowry [6], using the zinc enzyme as standard. The same extinction coefficient was found for the blue and for the native enzyme as long as the cupric enzyme retained its blue color. The activity of the cupric enzyme was OS%-1% that of the native zinc enzyme. In this vahre, the lower occupancy of the catalytic metal binding sites in the cupric enzyme is not taken into account. Since the H4Zn(n)senzyme shows residual activity between 0% and OS%, it cannot be decided definitely whether the cupric enzyme is to be regarded as inactive or slightly active. Whereas the crystals retained their color on standing under nitrogen, the solution of the cupric enzyme turned almost colorless under a period of 24 h. The activity of this colorless enzyme increased to about eight percent. (This would amount to about 20% in a fully substituted cuprous alcohol dehydrogenase). The colorless enzyme could be reconverted to a blue species upon addition of potassium hexacyanoferrate(II1). We interpret the change of color and activity of the cupric enzyme as being due to an intramolecular redox reaction yielding the cuprous enzyme. Possibly, a disulfide bridge is formed between cysteines 46 and 174. This would enable the cuprous ion to remain bound in the catalytic site. CompIexes of Cul* with disuhides are well known [7] _ However, the stoichiometry of this reaction remains unclear, since the reduction of the cupric ion requires one electron, whereas the formation of the disulfide bridge yields two electrons. As illustrated in figure I, the conversion of the cupric into the cuprous ion is accompanied by the occurrence of LMCT transitions in the ultraviolet spectrum of the enzyme below 300 nm similar to those observed in copper metallothioneine [S] _The optical
and esr spectra
of the cupric enzyme
are shown in figures 2 and 3 (upper trace)_
In Tables I and 2 the parameters of optic& and esr spectra are given, together with relevant data from naturally occurring blue copper proteins. These data show convincingly, that the state of the copper in the blue Cu(c)o_eZn(n)1_8-enzyme can be described as similar to Type 1 Cu 2+ of the blue copper proteins [9] _The visible spectrum of the blue Cu(c) o.eZn(n)l.aenzyme is similar to that of azurin (see Table 1) [lo], while the esr spectra indicate that the cupric enzyme appears to be similar to stehacyanin [9] , rusticyanin [ 111 and plantacyanin [12] due to the occurrence of rhombic components in the esr spectrum (see figure 3, upper trace)_ These components are almost absent in plastocyanin, azurin, and umecyanin [9, 131. Furthermore in the cupric enzyme Iigand hyperfine structure (most probably due to His 67) is observed in the esr spectrum (see figure 3, upper trace)_ When measuring the esr spectra of the unhganded cupric enzyme it was observed that upon cooling to 77 K the bIue color changed to violet. The optical spectrum of the cupric enzyme at 77 K is shown in figure 4 (solid line). The low-energy band is better resoIved at 77 K; the main band is blue-shifted 40 nm upon cooling. The blue color reappears again on warming to 296 K. A detailed analysis of this temperature dependence has not yet been made.
Wolfgang Maret et aL
244
FIGURE L Ultraviolet eIectronic absorption spectra of cupric (right) and cuprous (left) horse liver alcohol dekydrogenase in 25 mM TesjNa+, pE 69_ Concentration of enzyme: 8.3 & (right) and 26 pM (left).
Effect of Coenzyme and Inhiiitor on the Spectra of Cu(c)o_eZn(n)I_a-Horse Alcohol ikhydmgenase Lie Binary Complexes with NAP
Liver
or &razo!e
a Oxidked coenzymk Binding of NAD+ causes a large redshift of the charge transfer band from 620 to 650 mn at 296 K and from 580 to 595 mn at 77 K (Figure 5, solid line and Figure 7, solid line). This resembles the behavior of the Co(c)aZn(n)senzyme [14] - Since the coenzyme is not coordinated directly to the catalytic metal ion [2], a conformation change is likely to occur which affects the geometry of the catalytic metal binding site. Also, the esr spectrum (Figure 6, upper trace) indicates some change occurring in the metal binding site. The superhyperfme interaction of nitrogen is much less resolved upon binding of the coenzyme. However, the data in figures $6, and 7 and Tables I and 2 showthat even in the binary complex with NAD+ the state of the copper can still be described as Type 1 Cu*+. b. Pymzoie: This ligand acts as a competitive inhibitor of ethanol oxidation and coordinates directly to the catalytic zinc ion in the native enzyme [ 15]_ In the absence of coenzyme, the esrspectrum becomes axial with concomitant increase of Ai from 30 to 115 X 10m4 cm-l. This indicates a change of the state of the copper from its Type I FIGURE 2. Visiile electronic absorption spectrum of cupric horse liver alcohol dehydrogenasein 25 mM Tes/Na+, pH 6.9 at 296 K. Concentration of enzyme: 630 pIK
Copper(D)
Horse Liver Alcohol Dehydrogenase
245
.___‘._A_‘_.
2600
k__
ax!0
.
_.
3.00
G
FIGURE 3. esr spectra of native cupric horse liver alcohol dehydrogenase (upper trace) and of its binary complex with pyrazole (lower trace) recorded at X-band frequency. Temperature: PO0 K; concentration of enayrne: 630 &f in 25 rnM Tes/Na*, pH 6.9; concentration of pyrazole: 40 mh4.
upon binding of pyrazole. Ligand byperfime structure (AIN = 14 X 10-a cm-l) due to two nonequivalent nitrogen nuclei (i.e., the imidazole of His 67 and of pyrazole) is observed in the go region of the esr spectrum (Figure 3, lower trace). Upon binding of pyrazole to the enzyme a marked acceleration of the reduction of the cupric ion is observed at 296 K. Therefore the intensity of the bands in the visible spectrum of the binary complex could not be measured accurately. No shift of the 620 run band was observed at 296 K. On cooling to 77 K;the color of the enzyme changes from blue to pink (Figure 4, broken line). When the binary com- plex was cooled immediately after mixing of the cupric enzyme with pyrazole, the blue color reappeared on warming to 296 K. cJtaracter&ics
TABLE
1: Absorption Maxima of Cupric Horse Liver Alcohol Dehydrogenase and its Complexes in Comparison with Azurin and StellacyanirP A,,,&m)
&lYU (nm) at 296 Kc 1 native enzyme 2 binary complex with NAB+ 3 binary complex with pyrazole 4 ternary complex with pyrazole and NAD+ s azurin 6 stellacyanin
377 442 (E = 600) (E = 500)
620 (E = 2000)6
at 77 Kc
365 430 580 755
435
650
365 420 595
387
432
620
405 495 690
390
460
550
690
563
627
385 460 535 680
480
(E = 185) (E = 504)
443 560 (E = 942) (E = 1542)
(e = 3798) 603 (E = 3549)
u Taken from Ref. 10. b Extrapolated to one g-atom Cu*+ per subunit on the basis of both the experimentally determined metal content and the protein coneentratioa c Values of e givenin (M-1 cm-l).
Wolfgang Maret et aL
246
TABLE 2t Spin-Hamilt~nianParametersof Cupric Horse Liver Alcohol Dehydrogenaseand its Complexes. For Comparison the Data of Azurin, Stellacya.ni+ and a Low Molecular Weight Compoundb are also Listed
1 native cupricenzyme 2 binary complex with NAD+ 3 ternary complex with NAD+ and pyrazok ‘4 steuacyanin 5 z3.zoG 6 binary complex with 7
32 36 56
pyrazole
115
bM35dimethylpyxazole-lcarbodithioato)coppe~~
160
Data taken from Ret 9b Data taken from Ref. 24. C Values of A givenin ( lW4
14
30 32
56 7
15
224 2.29 226
208 206
14
2.20
205
15
216
29 7
15
2.25 2-24
2.03
2.04
2.07
0
cm-l).
The Ternary CompItx with NAD’ antipymZoIe. The native zinc enzyme forms strong ternary complexes with reduced coenzyme and fatty acid amides or with oxidized coenzyme and pyrazole [1] _ In the enzyme reconstituted with cobalt in the catalytic site the same binary and ternary complexes are also formed j14]. In the cupric enzyme we studied only the ternary complex formed upon binding of NAIY to the binary enzyme-pyrazole complex. The esr measurements (Figure 6, lower trace) indicate that the state of the copper changes again from the novel type to Type 1 Cu2+. The ternary complex is blue at 296 and 77 K (Figure 5, dotted line and Figure 7, broken line)_ Thus, ternary complex formation seems to stabilize the Type 1 state of the catalytic cupric ion within a wide range of temperature_
FIGURE 4. Viiile electronicabsorptionspectraof cuprichorseliveralcohol dehydrogenase(solid line) and its binary compkx with pyrazole (broken line) in 25 mM T-a+, pH 6.9 at 77 K; concentrationof enzyme: 630 /.M; concentrationof pyrazole: 40 mM.
CoPPerfII) Horse Liver Alcohol
Dehydrogenase
az-
u-
I UIO
I 500
I 600
I 700
I 800
hhnl FIGURE 5. Visible electronic absorption spectra of cupric horse liver alcohol dehydrogenase in its binary complex with NAD+ (solid line) and ternary complex with NAD+ and pyrazole (dotted line) in 25 mM Tes/Na*, pH 6.9 at 296 K; concentration of enzyme: 109 uM; concentration of NAW: 632 mM; concentration of pyrazoler 9.9 mM.
8
‘
2600
3000
I
3Lal
1
G
FIGURE 6. esr spectra of cupric horse liver alcohol dehydrogenase in its binary complex with
. . NAD+ (upper trace) and ternary complex with NAD* and pyrazole (lower trace) recorded at X-band frequency; temperature: 100 K; concentration of enzyme: 630 /.&f in 25 mM Tes/Na+, pH 6.9; concentration of NAD+: 2.3 n&f; concentration of pyrazole: 3.8 mM.
Wolfga_ng Maret et aL
248
FIGURE 7. Visiile electronic absorption spectra of cupric horse liver alcohol dehydrogenase in its binary complex with NAD+ (solid line) and temary complex with NAD* and pyrazole(broken line) in 2.5 mM T.es/Na*, pH 6.9 at 77 K; concentration of enzyme: 630 /.&f; concentration of NAD*r 2.3 rri!!; concentration of pyrazole: 3.8 mM.
Coxnparisonof the Binding Sites of the Blue Copper Proteins and Alcohol Deplydrogenase An important difference between the genuine Type 1 copper proteins and the cupric enzyme is the occurrence of mo cysteine residues as metal ligands in the latter. This probably is the reason for the slow conversion into a cuprous enzyme. In the genuine Type 1 copper proteins nature has elegantly circumvented the possible formation of a dimbide bridge within the metal binding site by choosing cysteine and methionine as metal ligands together with two histidine residues, as has been found in plastocyanin and azurin [3,16] _ The conserved tetrahedral binding site upon removal of the catalytic zinc ion would not only provide the proper environment for the Cua+ ion to render a Type 1 Cu2+ center but also would offer the most favorable site for Cul+ ions that prefer tetrahedral complexes. In this context, the enhanced enzymatic activity of the cuprous alcohol dehydrogenase is remarkable_ This was observed after an internal oxidation-reduction process which yielded cul+ in the catalytic site and possibly a disulfide bridge between Cys46 andCys 174. Cleariy, the preparation and examination of a cuprous enzyme built up from Cul+ and the H4Zn(n)2enzyme is highly desirable.
Binding of PyrazoIe Creates a Novei Type of Cu2+ Protein As in the native horse liver alcohol dehydrogenase and the cobalt substituted enzyme the cupric Cu(c),-,GZn(n)I_8enzyrne binds pyrazoIe by coordination to the catalytic metal ion. In the native Type 1 Cu2’ proteins no binding of inner sphere ligands has been observed and the artificial cupric enzyme seems to be unique iu this respect_ The binding of pyrazole to form the binary complex changes the state of the cupric ion:
Copper(D) Horse Liver Alcohol Dehydrogenase
249
The data in Tables I and 2 demonstrate that the optical and esr properties of both the free cupric enzyme and of its ternary complex are similar to those observed for Type 1 Cu2+ proteins. In the binary complex with pyrazole, the optical and esr properties are not in accordance with any of the established states of Cu2+ in copper proteins_ Sakaguchi and Addison point out in a recent article [17] that the quotient g”/Al may be used as “a convenient empirical index of distortion of the donor set from planar toward tetrahedral.” In the unliganded cupric horse liver alcohol dehydrogenase the value gn/Alt = 750 cm is in accordance with those found for the Type 1 copper proteins_ In contrast, a value gH/Aa = 147 cm is found for the binary complex of the cupric enzyme with pyrazole. This ratio is characteristic of Type 2 Cu2+. However, the optical spectrum exhibits the intense charge-transfer characteristically found only in Type 1 Cu2* proteins. Also, the binary complex with pyrazole does not bind fluoride ions which has been regarded as a criterion of Type 2 Cuz+ sites [ 18 ] . It should be emphasized that the pyrazole complex of the cupric enzyme is chemically more similar to plastocyanin and azurin than the unliganded cupric enzyme, since the copper is bound to two sulfur and two nitrogen atoms. The different state of the cupric ion in the binary complex might therefore be ascribed to differences in the coordination geometry only, if we assume that pyrazole substitutes the metal bound water molecule. A nearly square planar arrangement seems reasonable for this species (cf. the gll/All values for the binary complex and the model 7).
The Catalytic Copper Ion, Although inactive, is a SensitiveReporter of Events Related to the Catalytic Cycle From numerous structural and kinetic studies on the native zinc alcohol dehydrogenase it is well known that binding of the coenzyme induces a major conformational transition in the protein prior to the binding of the substrate [l] _As a consequence of this transition, the catalytic metal ion becomes able to bind and to activate the substrate. Additional support for this sequence of events has been obtained from spectroscopic studies using active site-specific reconstituted cobalt alcohol dehydrogenase [ 19]_ Therefore it is promising to observe that the binding of NAD+ (and of NADH, unpublished results) to the copper enzyme alone gives rise to changes in both the optical and esr spectra which must be interpreted as distortions of the coordination sphere. A different set of spectra is obtained in the ternary complex with NAD+ and pyrazole. In both complexes, the state of the copper can be described as Type 1 Cu2+_ This means that in the framework of this coordination type the conformation changes induced by the coenzyme and subsequent formation of ternary complex with inhibitor are accompanied by certain changes in the coordination sphere of the catalytic cupric ion in terms of bond angles and probably also bond distances. In view of the negligible activity shown by the cupric enzyme it may be suspected that the coenzyme induced transitions in the protein structure are rather independent of the kind of metal present in the catalytic site. The metal seems to be generally involved in this conformation change, irrespective of its ability to activate the substrate. Evolutionary Aspects The similarity between the catalytic site in horse liver alcohol dehydrogenase substituted with Cu2+ ions and the copper binding site of certain blue copper proteins
WoIfgang Maret et al.
250
leads to the question of whether the catalytic zinc binding site of alcohol dehydrogenase and the copper binding sites of the blue copper proteins may share some common ancestors_ Common structural features observed in both classes of proteins are the occurrence of suIfur as metal ligapd and as far as available data reveal a distorted tetrahedral coordination geometry [3, 163 _The metal atoms in both plastocyanin and horse liver alcohol dehydrogenase are accessiile from two directions; one access is provided through a hydrophobic channel, the other one from a more hydrophilic environment, that is, the solvent in the case of plastocyanin and the anion binding site in the coenzyme binding domain of alcohol dehydrogenase. On the other hand, an important difference is obvious: The copper ion in plastocyanin is coordinated to four protein side- chains (Cys, Met, 2 His [3]) whereas alcohol dehydrogenase has three ligands from the protein (2 Cys, His [I]) and an exchangeable water molecule as fourth ligand: The participation of the metal in catalysis as it has been inferred from numerous studies seems to proceed via outer sphere interactions in the redox processes catalyzed by plastocyanin [3] and via direct coordination of the substrate to the catalytic metal ion in alcohol dehydrogenase [I91 _ Also, comparison of the tertiary structure of plastocyanin with the catalytic domain in horse liver alcohol dehydrogenase shows no obvious similarity_ This is in contrast to the folding of the polypeptide chain around the noncatalytic metal ion in horse liver alcohol dehydrogenase, which is similar to ferredoxin [2] _ It has already been pointed out that the catalytic metal binding sites of several zinc-containing enzymes including horse liver alcohol dehydrogenase show common features with respect to ligand composition and coordination geometry [20] _ Most probably, this is due to very general chemical principles governing the design of these zinc-binding sites rather than to evolutionary relationships_ In the particular case of alcohol dehydrogenase we believe that the occurrence of cysteine as ligand of the catalytic zinc in alcohol dehydrogenase, which seems to be a common property in this class of enzymes [21], is also dictated by the chemical reasons inherent in the catalytic mechanism and not by evolutionary relationships to blue copper proteins_ CONCLUSIONS The artificial copper protein Cu(c)zZn(n)z-horse liver alcohol dehydrogenase is a versatile biological model system for studying the unusual spectroscopic properties of native copper proteins. Moreover it is a flexible coordinative system that changes its coordination sphere upon interaction with ligands or due to conformational changes within the protein triggered by coenzymes. The redox behavior of copper(H) liganded to thiolate sulfur is another aspect to be studied with this cupric enzyme. The cupric liver alcohol dehydrogenase liganded with pyrazole has to be considered as a novel, probably planar type combining the esr characteristics of Type 2 and the intense absorption spectra of Type 1, although blueshifted. Thus square planar coordination wi’J1 sulfur ligands does not necessarily give rise to Type 1 behavior in terms of esr parameters and to Type 2 in terms of optical spectra. From our data pentacoordination, which has been proposed for the blue copper proteins [22] (and which is the most adequate description for Type 2 Cu2* [23]), for either the native cupric enzyme or its complexes cannot be exckded. The conserved tetrahedral geometry in the cupric enzyme and its complexes with coenzyme as compared to the native zinc enzyme demonstrates the usefulness of Cua+ as a reporter group. The Cu2+ is affected by the coenzyme triggered conformation
Copper(H)
Horse Liver Alcohol Dehydrogenase
251
change of the protein, which makes it possible to study details of the catalytic cycle even in the copper substituted species. Financialsupporr was obtained from Deutsche Forschungsgemeinschaft(Ze 1.5216) and For& der
Chemirchen liuiustrie. We appreciate discussions withProfessorsI. Bertini, W. Haase, H. Korbwsk< T. D. Luckey, M. M&&n,
G. Rot&,
and R. J. P. Willtims
REFERENCES 1. C.-I. Br%rd&, H. Jiimvall, H. Eklund, and B. Furugren, in The Enzymes. 3rd ed,. P. D. Boyer, Ed., Academic Press, New York and London, 1975, VoL XI, Chap. 3, p_ 103-190. 2. H.Ekhmd, B. Nordstrcm, E. Zeppezauer, G. Siiderlund, I. Ohlsson, T. Boiwe, B.-O. Soderberg, 0. Tapia, C-1. Bed&, and A. Akeson, Three-dimensional Structure of Horse Liver Alcohol Dehydrogenasc at 2.4 8, Resolution,L Mol. Biol. 102,27-59 (1976). 3. P. M. Coleman, H_ C. Freeman, J_ M. Guss, M. Murata, V. A. Norris, J. A. M. Ramshaw, and M. P. Venkatappa, X-ray Crystal Structure Analysis of Plastocyanin at 2.7 A Resolution, Nature 272,319-324 (1978). 4. W. Maret, I. Andersson, H. Dietrich, H. Schneider-BernRihr. R. Einarsson, and M. Zeppezauer. Site-Specific Substituted Cobalt(H) Horse Liver Alcohol Dehydrogenases, Eur. J. Biochem. 98,501-512 (1979). 5. K. Dalziel, The Assay and Specific Activity of Crystalline Alcohol Dehydrogenase of Horse Liver,Acta Chem. &and. 11,397-398 (1957). 6. 0. H. Lowry, N. 3. Rosenbrough, A. L. Farr, and R. J. Randall, Protein Measurement with the Folin Reagent,J. BioL c7rem. 193,265275 (1951). 7_ R &terberg. Models for Copper-Protein Interaction based on Solution and Crystal Structure Studies, Coord. Uzem. Rev. 12,309-347 (1974). 8_ H- Rupp and U. Weser, Conversion of Metallothionein into Cu-Thionein, the possible low molecular weight form of neonatal Hepatic Mitochondrocuprein, FEBS Lett. 44, 293-297 (1974). 9. A. S_ Brill. nansition Metals in Biochemisrry. Springer Veriag. Berlin and New York, 1977. 10. E. I. Solomon, J. W. Hare, and H. B. Gray, Spectroscopic Studies and a Structural Model for Blue Copper Centers in Proteins, Proc NatL Acad. Sci USA 73,1389-1393 (1976). 11. J. C. COX, R. Aasa and B. G. MahstrGm, EPR Studies on the Blue Copper Protein Rusticyanin, FEBSLett. 93,157-160 (1978). 12_ V- T. Aikazyarr and R. M. Nalbandyan, Piantacyanin from Spinach, FEBS Lett. S&272-274 (1975). 13. T. Stigbrand, B. G. Mahnstr~m, and T. V&nqard, On the State of Copper in the Blue Protein Umecyanin,FEBS Lett. 12,260-262 (1971). 14. H. Dietrich. W. Maret, 1. Andersson, and M. Zeppeaauer, Site-Specifically Substituted Cobalt Horse Liver Alcohol Dehydrogenase, Hoppe Seyler’s Z. Physiol. Chem. 359,1074 (1978). 15. H. Eklund. personal communication. 16. E. T. Edman, R. E. Stenkamp, L. L. Sicker, and L. H. Jensen, A Crystallographic Model for Azurin at 3 A Resolution,J. MoL B&L 123,3547 (1978). 17. U. Sakaguchiand A. W. Addison. Spectroscopicand Redox Studies of Some Copper(H) Complexes with Biomimetic Donor Atoms: Implications for Protein Copper Centers, J. cherry. SosocDa&on 1979,600-608. Cnemistry, R. J. P. Williams and J. R. R. F. 18. B. G. Malmstriim, in New ‘12ends in Bihnoaanic Da Silva, Eds., Academic Press, London and New York, 1978,Chap. 3. 19. H. Dietrich, W. Maret, L. Wall&, and M. Zeppezauer, Active SiteSpecific Reconstituted Cobalt@Il Horse Liver Alcohol Dehydrogenase: Changes of the Spectra of the Substrate Trans4N,l+Dlmethylaminocinnam aldehyde ar@ of the Catalytic Cobalt Ion Upon Ternary Complex Formation with NADH and 1,4,5,6-Tetrahydronicotinamide Adenirre Dinucleotide, Eur. L B-em., 10,267-270 (1979).
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20. P. Argos, R_ M- Gravito. W- Eventoff, bi. G. Rossmann.and C-1. BrZnde’n,SimilaritiesIn Active Site Center Geometries of Zinc-containingEnzymes, Proteasesand Dehydrogenases,1. MOLBioL 126.141-158 (1978). 21. H_ JamvaU, Differences between Alcohol Dehydrogenases,Eur. J. Biocitem 72. 443-452 (1971). 22 B. Mondovi, L. Morpurgo, G. Rotilio and k Finazzi-A&, in Iron and Copper J+ot&s, K. T. Yasunobu, H. F. Mower, and 0. Hayaishi,JZds.,A&. Exp. Med. BioL 74,424-437 (1976). 23. G. RotiIio, L. Morpurgo, L. Calabrese, A. Finazi-Agr& and B. Mondovi, in Metal-Lignnd Interactiims in Organic Chemimy rmd Biochemisrry. B. Pulbrmnand N. GoIdbIum Eds., D. Reidel PublishingCompany, DordrechtHolland, part I, p. 243-253 (1977). 24. R. D. Bereman,G. D. Shiekls,and D_ NaIewajek,Characterizationof the CoordinationProperties of Bis(3,S-dimethyIpyrazoI~l~bodithioato) copper(ID, Inog. C7zem 17,3713-3714 (1978).
ReceivedJwze II. 1979; revisedOctober 16.1979.
’