The reaction of nitric oxide with copper proteins and the photodissociation of copper-NO complexes

38

Biochimica et Biophysica Acta 916 (1987) 38-47

Elsevier BBA 32966

T h e reaction o f nitric o x i d e with c o p p e r p r o t e i n s and the p h o t o d i s s o c i a t i o n of c o p p e r - N O c o m p l e x e s A . C . F . G o r r e n , E. d e B o e r a n d R . W e v e r Laboratory of Biochemistry, University of Amsterdam, Amsterdam (The Netherlands)

(Received 15 April 1987)

Key words: Copper protein; Nitric oxide; Photodissociation;Absorbance, low temperature; ESR

The reactivity with nitric oxide was investigated for a number of type-l, type-2 and type-3 copper proteins: azurin from Pseudomonas aemginosa (type-1 copper); bovine superoxide dismutase, diamine oxidase from pig kidney and galactose oxidase from Dactylium dendroides (type-2 copper); haemocyanin from Helix pomatia (type-3 copper); the blue oxidases ceruloplasmin from pig serum, and ascorbate oxidase from Cucurbita pepo meduUosa. Type-1 copper formed complexes with NO in the oxidised state, which complexes were only fully formed at low temperatures and could be photodissociated at 77 IL Complex formation led to the disappearance of the EPR signal of type-1 copper and of the optical absorbance band in the 600 nm region. In azurin, pbotodissociation caused the reappearance of the original 625 um absorbance band, but in the blue oxidases, a new band with lower intensity was found at 595 nm instead of the original absorbance band at 610 nm. In all cases, the EPR signal of type-1 copper did not return. These results are best explained by the formation of a photolabile type-1 CuI+-NO + complex. They also indicate that in the complex formed, the type-1 copper structure is probably not disrupted, and that after illumination, the nitric oxide molecule is still in the near vicinity of the copper atom. Type-2 copper did not react at all with nitric oxide, and type-3 copper formed complexes with nitric oxide in both the oxidised and the reduced state, but photodissociation of these complexes could not be demonstrated.

Introduction In copper proteins (see for reviews concerning active sites Refs. 1 and 2), three types of copper can be distinguished. Type-l, or 'blue' copper, is characterised by an unusually strong optical absorbance in the 600 nm region and an EPR signal with a small hyperfine splitting. It is present in a few small proteins (for instance plastocyanin, azurin and steUacyanin) that are usually involved in redox reactions, and also, together with type-2 and type-3 copper, in the blue oxidases ceruloplasmin, ascorbate oxidase and laccase. In plastoCorrespondence: R. Wever, University of Amsterdam, P.O. Box 20151, 1000 HD Amsterdam, The Netherlands.

cyanin and azurin, the type-1 copper structure has been determined in detail. In these proteins, copper ligates to two nitrogen atoms of histidines, one sulphur from a methionine, and one sulphur from a cysteine. The ligation of the copper to the thiol group of the cysteine and the highly distorted tetrahedral geometry around the copper ion are thought to be the cause of its unusual spectral properties. Type-2, or 'normal' copper, is by far the most abundant in nature. Its spectroscopic properties are similar to those found in inorganic copper complexes and feature an EPR-signal with a larger hyperfine splitting and no strong optical absorbance. Type-2 copper proteins, in contrast to type-1

0167-4838/87/$03.50 © 1987 Elsevier SciencePublishers B.V. (Biomedical Division)

39 and type-3 copper proteins, do not form a homogeneous group, but are very variable in structure, function and properties. Their geometry is usually square-planar. Type-3 coppers exist in pairs. They are present in the proteins haemocyanin and tyrosinase and in the blue oxidases. Their optical properties differ from one another: in the blue oxidases, they are thought to be responsible for a weak absorbance band at 330 nm. In oxyhaemocyanin and deoxyhaemocyanin, the reduced copper sites show a moderately strong absorbance band at 570 nm and an intense band at 345 nm. Type-3 copper pairs are EPR-silent, either due to an antiferromagnetic exchange between the two copper nuclei, or because the copper ions are in the cuprous state. Nitric oxide reacts with the copper atoms in some copper proteins. This was first demonstrated with haemocyanin from Helix pomatia [3-6], later also with tyrosinase [7]. It was shown that deoxyhaemocyanin can be rapidly oxidised by N O to yield methaemocyanin, which then, in a slow reaction, will bind one nitric oxide molecule per copper pair [6]. Nitric oxide also reacts with the copper atoms of the blue oxidases ceruloplasmin [8-10], ascorbate oxidase [11] and laccase [12,13], but as these proteins contain all three types of copper, the interpretation of the observations is rather complicated. So, it could happen that the reactions of fungal and tree laccase on the one hand, and ceruloplasmin and ascorbate oxidase on the other, have been interpreted quite differently, although in many respects their reactions seem to be similar. The results obtained with ceruloplasmin [8-10] and ascorbate oxidase [11] were explained by assuming the formation of a stable complex of oxidised type-1 copper with N O and the binding of N O to reduced type-3 copper (two NO molecules per copper pair); oxidised type-3 copper also reacts with nitric oxide, but it is not clear whether N O binds to oxidised type-3 copper, or whether the type-3 copper site is first reduced by NO. On a larger time scale, also type-1 copper is reduced. Type-2 copper does not react with NO. Martin et al. [13], who studied the NO-reaction with tree and fungal laccase, reached different conclusions. In the fungal enzyme, all copper

atoms are reduced by NO; type-2 copper more slowly than the other copper atoms. N O was also assumed to form a complex with reduced type-2 copper. The tree enzyme is also reduced by NO, but very slowly. At the same time, the reduced coppers were supposed to be slowly oxidised by NO. Nitric oxide binding was proposed to occur only with the reduced enzyme, with type-2 copper and type-3 copper (one N O per copper pair). Finally, nitric oxide has been shown to react with CuB (but not with CUA) in cytochrome c oxidase [14]. Nitric oxide forms a complex with oxidised Cu B that can be photodissociated at low temperatures [15]. To establish whether this light sensitivity is a specific feature of the cytochrome c oxidase oxygen-binding site or whether it is a general property of Cu. NO complexes, we have investigated the effects of addition of nitric oxide and illumination on several copper proteins. These studies may also help in interpreting the reactions of NO with the blue oxidases. We have studied the reaction of N O with and the effect of illumination on the type-1 copper protein, azurin, from Pseudomonas aeruginosa, which contains one Cu atom per molecule. Of the type-2 copper proteins, we selected the enzymes superoxide dismutase, diamine oxidase and galactose oxidase. We also studied the effect of illumination on NO-incubated haemocyanin from H. pomatia, one of the copper proteins containing type-3 copper. Finally, we investigated the photosensitivity of the NO complexes with the multi-copper protein ascorbate oxidase and extended our previous work on ceruloplasmin [16]. Ascorbate oxidase from zucchini squash, Curcurbita pepo medullosa, contains three type-1 copper atoms, one type-2 copper atom and two type-3 copper pairs [11]. Ceruloplasmin from pig serum contains two type-1 copper atoms, one type-2 copper atom, and two type-3 copper pairs [1,2]. The laccases, which are not studied here, contain one type-1 copper atom, one type-2 copper atom and one type-3 copper pair [1,21. Materials and Methods

Ceruloplasmin was isolated from pig serum according to Ref. 17. The A610 nm/A28o nm ratio of

40 the preparations was 0.043-0.046. The concentration was calculated from the absorbance coefficient of 10.9 mM - 1 . cm -a at 610 nm [18]. Ascorbate oxidase, a gift from Prof. B. Mondovi (Rome), was purified from green zucchini squash (C. pepo medullosa), according to Ref. 19. The absorbance coefficient at 610 nm (9.6 mM -~cm -1) was used to determine the protein concentration [20]. Haemocyanin was prepared from H. pomatia [21]; the protein concentration was determined according to Ref. 22. Azurin from P. aeruginosa, bovine superoxide dismutase, diamine oxidase from pig kidney, and galactose oxidase from Dactylium dendroides were purchased from Sigma. As judged from the spectroscopic data, these proteins were pure, except for diamine oxidase which contained some contaminations. For azurin, the A625 nm/a280 n m ratio was 0.52. Experiments using ceruloplasmin, ascorbate oxidase, azurin and haemocyanin were performed in 100 mM sodium acetate (pH 5.5). Superoxide dismutase, diamine oxidase and galactose oxidase were dissolved in 100 mM potassium phosphate (pH 7.4). Nitric oxide was obtained from Matheson Gas Products. Anaerobiosis was carried out as described in Ref. 23. Low-temperature optical experiments were carfled out either in an Aminco-Chance DW-2 spectrophotometer as reported by Wever et al. [24], or in an Oxford Instruments 204-CA cryostat that was placed inside a modified Cary-14 spectrophotometer [25]. The samples were anaerobically transferred from a tube fitted with a septum via a gas-tight Hamilton syringe to an optical cell with perspex windows and immediately frozen in liquid nitrogen when the Aminco-Chance DW-2 spectrophotometer was used, or the samples were transferred to a plastic cuvette (1 cm), that was kept in a nitrogen atmosphere, and subsequently frozen in the cryostat. In the latter case, and also in experiments in which the light intensity was varied, the samples were prepared in 70% glycerol ( v / v ) to acquire transparency. Optical experiments at room temperature were performed with Thunberg cuvettes in a HewlettPackard 8451A diode array spectrophotometer. EPR-spectra were measured with a Varian E-3 spectrometer at 77 K and with a Varian E-9

spectrometer at lower temperatures; the experiments were performed in Thunberg-type anaerobic EPR tubes. The incubation time with NO varied from 2 to 4 rain in the EPR and room-temperature optical experiments. For the low-temperature optical experiments, the incubation time was 10 to 15 rain. Illumination was achieved by using one of the following lamps: a 500 W slide projector, a 150 W continuous xenon lamp (Oriel) or, where indicated, a 800 W mercury lamp. The light intensity could be varied with a calibrated set of neutral density filters (Oriel). Results

The absorbance spectra after the addition of nitric oxide to anaerobic ceruloplasmin and after subsequent illumination of the sample were measured. As was shown previously [8], incubation of 100 kPa N O with ceruloplasmin causes partial disappearance of the 610 nm absorbance band. When the absorbance spectrum is measured at a temperature of 77 K, the disappearance is almost complete, resulting in loss of the typical blue colour. Simultaneously, a small absorbance band at 390 nm appears on top of a broad absorbance increase in the lower wavelength region. When the sample was illuminated, it turned blue-green and a new absorbance band at 595 nm apepared (not shown, but see Fig. 1 of Ref. 16). At the same time, the 390 nm absorbance band decreased. The intensity of the 595 band reached a maximal level of only 35% compared to the original 610 nm band. (A weak band at 450 nm that had disappeared together with the 610 nm band also reappeared, but more slowly than the 595 nm band.) Prolonged or more intense illumination (using a 800 W mercury lamp) had no further effect on the absorbance spectrum. The effects of illumination were completely reversed when the sample was warmed to 140 K. This procedure could be repeated many times with the same sample. Fig. 1 shows the initial rate of formation of the 595 nm band as a function of the intensity of illumination. The linear relationship found here shows that we are dealing with a primary photoeffect.

41

1.2 el

f 0.8

b

f 0.4

0.0

I

o.oo

o.25

I

0.50 I/I o

I

0.7'5

I

1.oo

I

260

I

1 300

I

I 340

B (mT)

Fig. 1. Dependence of the rate of formation of the optical absorbance band at 595 nm in ceruloplasmin on the light intensity. The rates were calculated from the initial rate of the dissociation reaction. Conditions: 78 ~M ceruloplasmin, 100 mM sodium acetate (pH 5.5)/50% glycerol (v/v); 90 kPa NO; temperature, 77 K. For irradiation, white light from a 500 W slide projector was used. Nitric oxide was allowed to recombine with ceruloplasmin by warming the samples to 140 K, after which the sample was illuminated again.

Fig. 2. The effect of illumination on the EPR spectrum of ceruloplasmin in the presence of NO. (a) 174 /~M ceruloplasmin; (b) after addition of 55 kPa NO to (a); (c) after 17 min of illumination at 77 K of (b). The enzyme was dissolved in 100 m M sodium acetate (pH 5.5). EPR conditions: frequency, 9263 MHz; microwave power, 3.5 mW; modulation amplitude, 10 G; temperature, 77 K. Spectrum (a) was recorded at 0.63 x the gain of (b) and (c). Illumination was performed with a 500 W slide projector.

In the E P R spectrum at 77 K, anaerobic addition of N O to ceruloplasmin causes the disappearance of the type-1 copper signal, leaving only the type-2 copper signal, as was shown previously [8]. Surprisingly, illumination did not lead to the reappearance of the type-1 copper signal. The only effect of illumination that was observed was the rise of a four-line signal at g = 2.01 (Fig. 2). This signal could also be observed when ceruloplasmin (oxidised) was illuminated in the absence of NO, and was previously described by Henry and Peisach [26]. Reduced ceruloplasmin shows no bands in the optical absorbance spectrum, no E P R signal and it is not photosensitive. When the reduced enzyme is incubated with nitric oxide (55 kPa), the E P R spectrum (at a temperature of 15 K or lower) exhibits signals at g = 2 and g = 4 [9]. At g = 4, a similar, but not identical signal can also be generated by the addition of N O (90 kPa) to the oxidised enzyme [10]. Both signals have been ascribed to

type-3 Cu-NO complexes. We studied the effect of illumination on these signals and both signals turned out to be completely insensitive to light (not shown). Similar experiments were performed with the closely related enzyme ascorbate oxidase (Fig. 3). With ascorbate oxidase, anaerobic addition of N O also resulted in almost complete bleaching of the 610 nm band and of the blue colour, as was already reported by Van Leeuwen et al. [11]. Illumination again induced the appearance of a new band at 595 nm, while the sample turned bluegreen. In this case, though, the intensity of the new band was only 18% of the original 610 nm band. In the E P R spectrum, addition of N O brought about the disappearance of the type-1 copper signal, leaving the type-2 copper signal unaltered. As with ceruloplasmin, illumination did not induce reappearance of the type-1 copper signal, but only caused the formation of a narrow signal centered at g = 2.00 (Fig. 4).

42

A

I 450

I

I

I

I~""----

5,50

65O

h(nm) Fig. 3. Effect of light on the optical absorbance spectrum at 77 K of nitrosyl ascorbate oxidase. (a) 43/~M ascorbate oxidase; (b) after addition of NO (85 kPa) to (a); (c) after 50 rain of illumination at 77 K of (b). The enzyme was dissolved in 100 mM sodium acetate (pH 5.5). Illumination was performed with a 500 W slide projector.

Fig. 5 shows the effects of nitric oxide and illumination on the absorbance spectrum of azurin. When nitric oxide (100 kPa) was anaerobically added to azurin, the effect on the absorbance spectrum at room temperature was only small; the intensity of the 625 nm band decreased by about 15% (with the naked eye no colour change could be observed). However, when the temperature was lowered to 77 K, the sample decoloured and the 625 nm band nearly vanished. Illumination led to an almost full restoration (85%) of the 625 nm absorbance band. Azurin was more sensitive to light than ceruloplasmin and ascorbate oxidase: the NO-treated azurin had to be carefully kept in the dark to prevent the blue colour from returning. The light-induced process was completely reversible: upon warming the sample to about 140 K, the 625 nm band disappeared again. This procedure could be repeated many times. The decrease by 15% of the 625 nm band that was observed at room temperature was completely reversible: removal of NO resulted in re-formation

I

I

I

Q

u c-

/ l 260

t

I 300

B

I

e-i a

V

I

3t,0

(roT)

Fig. 4. The effect of light on the EPR spectrum of ascorbate oxidase in the presence of NO. (a) 43 txM ascorbate oxidase; (b) after addition of NO (83 kPa) to (a); after 15 min of illumination at 77 K of (b). The enzyme was dissolved in 100 mM sodium acetate (pH 5.5). EPR conditions: frequency, 9262 MI-Iz; microwave power, 35 roW; modulation amplitude, 10 G; temperature, 77 K. Spectrum (a) was recorded at 0.4 x the gain of (b) and (c). Illumination was performed with a 500 W slide projector.

500

,

,

600

700

w o v e l e n g t h (nm) Fig. 5. The effect of illumination on the optical absorbance spectrum of NO-incubated azurin at 80 K. (a) 57/~M azurin; (b) after addition of NO (100 kPa) to (a); after 10 rain of illumination at 80 K of (b). The enzyme was dissolved in 100 m M sodium acetate (pH 5.5)/'/0% glycerol (v/v). Illumination was performed with a 150 W xenon lamp.

43 of the band. However, part of the NO-induced decrease at 450 n m appeared to remain. Since it is conceivable that azurin is reduced by N O and is reoxidised by light, the effect of light on azurin reduced with sodium dithionite was also studied. Illumination of the reduced enzyme did not restore the 625 nm band. Fig. 6 shows the effect of N O and illumination on the EPR spectrum of azurin under anaerobic conditions at 77 K. Addition of N O resulted in the disappearance of the type-1 copper signal. Illumination had no effect on the EPR spectrum, even though the E P R sample did turn blue. Trace d of Fig. 6 shows the E P R spectrum after removal of the nitric oxide by repeated cycles of evacuation and flushing with helium. The type-1 copper E P R signal had fully returned, which would indicate that the nitric-oxide reaction is completely reversible. This reversibility could also be demonstrated in the optical experiments if they were performed at room temperature in Thunberg cuvettes (not shown). Nitric oxide was also added to several type-2

copper proteins. The EPR and optical absorbance spectra of superoxide dismutase, pig kidney diamine oxidase and galactose oxidase were not affected by the anaerobic addition of 100 kPa N O and incubation for 1 h (not shown). As an example of a type-3 copper protein, the reaction of nitric oxide with deoxyhaemocyanin from H. pomatia was studied. This reaction was investigated previously [2-4] and takes place in two different steps. In the first step, nitric oxide acts as electron acceptor, resulting in the formation of methaemocyanin. This reaction is accompanied by the disappearance of the optical absorbance band at 570 nm and the formation of a broad signal at g = 4. In the second step, one nitric oxide molecule is thought to bind to each copper pair, which is accompanied by the appearance of a new EPR signal centred at g = 2. We investigated whether either of these two compounds was light sensitive. Illumination of deoxyhaemocyanin incubated with N O (85 kPa) had no effect on the optical absorbance and EPR spectra at 77 K and 15 K, regardless of the NO-incubation time was 3 to I5 min or 3 h (not shown). Discussion

220

L

250

I

300

I

350

t. O0

B (mT)

Fig. 6. The effect of light on the EPR spectrum of azurin in the presence of NO. (a) 375 ttM azurin; (b) after addition of NO (100 kPa) to (a); (c) after 10 min of illumination at 77 K of (b); (d) after removal of NO from (c). The enzyme was dissolved in 100 mM sodium acetate (pH 5.5). EPR conditions; frequency, 9130 MHz; microwavepower, 10 roW; modulation amplitude, 10 G; temperature, 77 K. Illumination was performed with a 150 W xenon lamp.

That nitric oxide reacts with copper proteins has already been shown for a number of these proteins: haemocyanin, ceruloplasmin, ascorbate oxidase, laccase and Cu B of cytochrome c oxidase are all reported to react with nitric oxide. The reactions can be divided in redox reactions on the one hand, and complex formations on the other. It is not always easy to decide whether one is dealing with a reduction of the copper atom or with the formation of a complex, as both will have the same or similar spectral consequences. This is especially true for the multi-copper proteins due to their complexity. Therefore, we decided to study the reactions of nitric oxide with proteins containing only one type of copper and we will discuss the results of those studies first. In the case of azurin, the vanishing of the 625 n m absorbance band and the g = 2 EPR signal can be due either to reduction or to complex formation. Several explanations can be considered. The copper atom might be reduced by nitric

44

oxide directly (model 1). It is also conceivable that N O reduces another group on the protein (designated as X), and that upon lowering the temperature to 77 K the electron is transferred to the copper atom, while upon illumination the electron is transferred back to the group X (model 2). Alternatively, nitric oxide might form a complex with the oxidised type-1 copper site (models 3 and 4). This complex formation could induce the observed effects on the optical absorbance and EPR spectra in two ways: binding of nitric oxide to the copper atom might disrupt the type-1 copper structure, causing the disappearance of the blue colour, while the disappearance of the EPR signal could be ascribed to an antiferromagnetic coupling of the electron spins of the copper atom and the NO molecule (model 3). On the other hand, the disappearance of the 625 nm band and the type-1 copper EPR signal could also be explained by the formation of a Cu+-NO + charge-transfer complex (model 4). Finally, we should consider the possibility that a group situated at the protein surface (designated X) binds the nitric oxide molecule, and that the observed spectral effects are due to intramolecular electron transfer (model 5). Cu 2+ ~

~Cu1+

NO

Cu2+X

(I)

NO +

~

)Cu2+

NO

low T X - . ~ Cul+X

(2)

h~,

NO, +

NO+Cu

2+ ~ - C u 2 + ' N o h ~ C u 2 +

NO+Cu

2+ # C u I + ' N O

NO+Cu

2+ . . X ~

+ ~Cu

2+ . . . . .

Cu 2+ . . X'NO

~

(3)

NO

.....

(4)

NO

C u 1+ . . . X . N O

+

hl,

~Cu

2+ . . . X . . N O

(5)

The photoeffect that we found rules out the simplest of these models, the reduction of Cu 2÷ to Cu 1÷ (model 1), because the reduced enzyme was not photosensitive. The other model in which N O only acts as an electron donor (model 2) can also be excluded, because of the reversibility of the N O reaction when N O was removed. Model 5 is also unlikely: with this model it is hard to imagine why

the EPR signal did not return upon illumination. Of the two remaining possibilities, model 3 is less likely, because at 77 K there is probably not enough thermal energy in the system to allow restoration of the disrupted type-1 structure. Therefore, we conclude that nitric oxide is able to bind to the type-1 copper site and that a Cu ÷N O ÷ complex is formed. There are two ways in which illumination would cause the effects observed by us: it could be a photodissociation (model a) or an electron transfer from reduced copper to N O ÷ (model b) C u 1 +. N O l + ~ C u z + . . N O

(a)

Cu I +.NO 1+h~cu2+.NO

(b)

Although model b cannot be ruled out on the basis of the evidence presented here, we are inclined to favour model a in analogy with the well-established photodissociation reactions of other NO complexes [27,28]. In order to explain the absence of a copper EPR signal in the illuminated samples, we have to assume that after photodissociation the nitric oxide molecule remains in the vicinity of the copper atom. We conclude that N O is able to form a complex with the oxidised azurin copper site and that this complex is photodissociable. The ability of azurin Cu 2+ to bind NO is all the more remarkable, as X-ray analysis has indicated that the shortest distance from the copper atom to the protein surface is 7.5 A [29]. We observed that at room temperature the 625 nm band decreased very little in the presence of nitric oxide, while at 80 K the band almost disappeared. This is in agreement with the expected behaviour of a complex, as dissociation constants generally decrease with decreasing temperature. About 15% of the 625 nm absorbance band was not restored by illumination. The loss of band intensity is caused by a slow reduction of the copper atom during the incubation with N O at room temperature. When the incubation time was kept short (less than 5 min), as was the case in the optical experiments at room temperature and in all EPR measurements, the reaction with N O was completely reversible.

45 It has previously been reported that the type-2 copper protein superoxide dismutase does not react with N O [8]. Our present results confirm this observation, and they also show that two other type-2 copper proteins, pig kidney diamine oxidase and galactose oxidase, were unable to react with nitric oxide. In contrast to the type-1 coppers, the type-2 coppers are not a homogeneous group, and one should therefore refrain from drawing too general conclusions. The reactions of nitric oxide with the type-3 copper protein haemocyanin have been reported by several investigators [2-4]. Those reactions were confirmed by us, but we were unable to detect any photoeffect. The results we obtained with the blue oxidases ceruloplasmin and ascorbate oxidase fit in well with the results discussed above. Type-2 coppers did not react with NO, type-3 coppers bound nitric oxide, but, even at 10 K, dissociation could not be demonstrated, neither with the reduced nor with the oxidised enzymes. The type-1 coppers of both enzymes reacted with NO. In analogy with the results we obtained with azurin, we assume that this reaction is the formation of a Cu2+-NO or Cul+-NO + complex. As in the case of azurin, complex formation is incomplete at room temperature: with 100 kPa of N O at 20°C, about 20% of the 610 nm absorbance band remains for ceruloplasmin [8], and about 65% for ascorbate oxidase [11]. The height of the 610 nm peak at room temperature depends on the concentration of NO rather than on the incubation time, which is a strong argument in favour of complex formation and against reduction. As with azurin, the complex could be photodissociated at low temperatures. The greatest difference with the azurin data is the fact that the band intensity after dissociation was much smaller than the intensity of the original band and that the peak position had shifted considerably to the blue after dissociation. While the original band was at 610 nm, the peak position after dissociation lay at 595 nm and the peak heights were 35 and 18% of the original band for ceruloplasmin and ascorbate oxidase, respectively. Two explanations are possible for this phenomenon. On the one hand, it maybe that the type-1 copper sites have

changed by the addition of NO, either as a result of the effect that NO-binding has on the type-1 copper sites themselves, or, indirectly, by effects due to the nitric oxide molecules that are bound at the type-3 copper sites. Such changes at the type-1 copper sites might manifest themselves by changes in the electronic properties, causing shifted peak positions and modified intensities in the optical absorbance spectrum. On the other hand, it is also possible that the different peak positions reflect intrinsically different type-1 copper sites. Ceruloplasmin contains two type-1 coppers, ascorbate oxidase three. If we allow for some reduction occurring during the incubation at room temperature, it could be that the bands we observed after dissociation originated from only one of the type-1 coppers in each case. That would mean that only one of the type-1 coppers forms a complex with NO that can be photodissociated at 77 K. As a consequence, this copper site would have an absorbance maximum at 595 nm. The other copper site(s), which would have an absorbance peak at higher wavelength, would either be reversibly reduced or have formed NO complexes that cannot be photodissociated at 77 K. An interesting detail is the observation that upon illumination, the weak 450 nm band was restored more slowly than the 595 nm band. This is reminiscent of the distinction Herv6 et al. [30] made between type-1 coppers that are strongly affected by anion binding, with an optical absorbance in the 500-700 nm region, and type 1 coppers that are weakly affected by anion binding, with an absorbance band at 450 nm. Van Leeuwen and Van Gelder [10] found that for nitric oxide the opposite is true: the 450 nm band is the one that is affected more by nitric oxide. Our observations confirm this. In his thesis [31] Van Leeuwen investigated the p H dependence of the optical absorbance spectrum of ceruloplasmin. The 450 nm band turned out to be strongly affected by pH, whereas the 610 nm band was not pH-dependent. On the basis of his results, he proposed that these pH effects were caused by protonation of one of the nitrogen atoms derived from a histidine residue. This suggests that the different behaviour of the 610 nm and 450 nm absorbancies could be caused by consecutive changes in the electronic

46 properties of the copper sites, rather than by different reactivities of different copper sites. Our observation that with azurin, which contains only one copper atom, the 625 nm and 450 n m absorbancies were also effected differently by nitric oxide support this interpretation. The reactions of nitric oxide with laccase as studied by Martin et al. [13] seem to be considerably more complicated than the reaction of N O with the other blue oxidases; redox reactions appear to play a more prominent role, especially after longer incubation times. For the fungal laccase, it seems that fast reduction of both type-1 and type-3 coppers and a somewhat slower type-2 copper reduction take place. After short incubation times, complex formation of N O with oxidised type-2 copper cannot be ruled out, but at least after 2 h of incubation, the enzyme is completely reduced. The tree enzyme exhibits very complicated behaviour when the incubation time has been long, and the observations made with the reduced enzyme are puzzling: but for short incubation periods the reaction of oxidised tree laccase with nitric oxide is similar to our results with the other multi-copper oxidases. More particularly, this is the case with the bleaching that occurred on freezing the sample, which Martin et al. [13] ascribe to reduction, but which we, in line with the reactions observed with azurin and the blue oxidases, interpret as complex formation of N O with type-1 copper. A comparison whould also be made with the copper atoms in cytochrome c oxidase and their reactivity towards NO. Cu A, which has been proposed by some authors [32,33] (but for a contrary view also see Refs. 34 and 35) to be structurally related to the type-1 copper proteins, to be totally inert to nitric oxide, and, in this respect, to resemble the type-2 coppers. Oxidised Cu B not only binds nitric oxide, but the complex is also photodissociable [15], like the type-1 copper N O complexes studied in this paper. This sets it apart from type-3 copper to which it is structurally related [36,37] and supports the claim made by some investigators [38] that Cu B has a type-1 structure. With regard to this issue, we should point out, however, that in order to decide whether or not a photoeffect occurs with type-3 copper, we had to rely heavily on EPR measurements; but our re-

sults obtained with type-1 copper atoms (restoration of the optical absorbance spectrum, but not of the EPR signal) have shown that EPR results might not always be a safe criterion. In this respect, it may be significant that also in the photodissociation experiments with the Cu2÷-NO complex, EPR signals from Cu 2÷ could never be detected, not even in situations where one would expect to see them (for instance in Fig. 2B of Ref. 39). Summarising, we reach the conclusion that the different types of copper react differently with nitric oxide. Type-1 coppers in the oxidised state formed complexes with nitric oxide that were photodissociable. On longer time scales, they tended to become reduced by NO. The type-2 coppers investigated by us did not react with nitric oxide. It should be remembered, however, that there is no such thing as a typical type-2 copper site, which leaves open the possibility that some type-2 copper sites might be reactive towards NO. Type-3 coppers showed rapid redox reactions with nitric oxide and could also bind NO; for those complexes no photodissociation could be demonstrated.

Acknowledgements The authors thank professor B.F. van Gelder for his interest and reading the manuscript, Mr. H. Dekker for assisting with the experiments, and Professor B. Mondovi for putting ascorbate oxidase at our disposal. This study was supported in part by grants from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) under auspices of the Netherlands Foundation for Chemical Research (S.O.N.).

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