er), three sharp, negative bands appear at 485, 442, and 340 nm with an equally intense ..... Solomon, E. I. (1981) in Copper Proteins, ed. Spiro, T. G. (Wiley,.
Proc. Natl. Acad. Sci. USA Vol. 82, pp. 3063-3067, May 1985
Chemistry
Low-temperature magnetic circular dichroism studies of native laccase: Spectroscopic evidence for exogenous ligand bridging at a trinuclear copper active site [coupled binuclear (type 3) copper/normal (type 2) copper/charge transfer transitions]
MARK D. ALLENDORF, DARLENE J. SPIRA, AND EDWARD I. SOLOMON* Department of Chemistry, Stanford University, Stanford, CA 94305
Communicated by John I. Brauman, December 21, 1984 L
The detailed nature of N- binding at the ABSTRACT multi-copper active site in native laccase is investigated through a combination of low-temperature magnetic circular dichroism (LTMCD) and absorption spectroscopies. This combination of techniques allows charge-transfer spectral features associated with N- binding to the paramagnetic type 2 Cu(II) to be differentiated from those associated with binding to the antiferromagnetically coupled, and therefore diamagnetic, binuclear type 3 Cu(II) site. Earlier absorption titration studies have indicated that N- binds with two different binding constants, yielding a high-affinity and a low-affinity form. The studies presented here are interpreted as strong evidence that low-affinity N- bridges the paramagnetic type 2 and diamagnetic type 3 binuclear Cu(ll) sites in fully oxidized laccase. This assignment is further supported by features in the MCD spectrum whose intensity correlates with an EPR signal associated with uncoupled type 3 Cu(ll) sites. In these sites, N- has displaced the endogenous bridge, thereby rendering the site paramagnetic and detectable by both LTMCD and EPR spectroscopy. High-affinity N- is found to bind to the paramagnetic type 2 Cu(ll) site in a limiited fraction of the protein molecules that contains reduced type 3 sites. Finally, the possible role of this trinuclear (type 2-type 3) Cu(ll) active site in enabling the irreversible reduction of dioxygen to water is considered.
I--,
CU 2
N~l-
CU +2
N"N
CU/2
CU+2
R
RL
HEMOCYANIN and TYROSINASE
LACCASE
FIG. 1. Comparison of exogenous ligand binding at coupled binuclear Cu(II) sites.
and tyrosinase, exogenous ligands are found not to bridge the type 3 site in T2D laccase (9, 10) (Fig. 1). In comparing exogenous ligand reactivity in T2D and native laccase, major differences have been observed (11). Whereas the fully reduced native enzyme is readily oxidized by 02, the binuclear Cu(I) site in T2D laccase is stable to aerobic oxidation (8, 12). In addition, N- binds to oxidized T2D laccase to generate a single N- -- Cu(II) charge-transfer (CT) transition at 450 nm (K 200 M-1, As 800 M-1lcm-1), while two N- molecules have been reported (11, 13) to bind to native laccase (K1 60,000 M-1 at 500 nm, Ae500 = 500 M-'-cm-', Ae410 = 630 M-l cm-l; K2 60 M-1 at 400 nm, AE 1900 M-1lcm-1). In earlier studies (11), it was proposed that the type 2 site indirectly stabilizes ligand binding at the type 3 site. Through low-temperature magnetic circular dichroism (LTMCD) spectroscopy, the nature of the interaction of exogenous ligands with the type 3 and type 2 centers can now be clearly defined. The new absorption features observed in N- reactions with native laccase have been associated (11, 13) with equatorial N- -+ Cu(II) CT. These transitions can only occur at the oxidized type 2 and type 3 centers, as the type 1 site in laccase is reasonably considered to contain no exchangeable positions. In order for a CT transition to be observed in the LTMCD spectrum, it should (i) have significant absorption intensity and (ii) be associated with a magnetically degenerate ground state. Two structural units exist in laccase from which N- -* Cu(II) CT bands may originate: an isolated, mononuclear type 2 Cu(II) and a binuclear type 3 Cu(II) pair. The first has a magnetically degenerate ground state with S = 1/2 , giving rise to EPR signals in the g 2.2 region. A ligand -- Cu(II) CT band originating from this ground state will have an associated MCD band whose intensity is proportional to 1/T, for kT >> gf3 H (C term). The second structural unit contains a pair of antiferromagnetically coupled S = 1/2 Cu(II) ions, with total spin states ST = 0,1, which are split by the exchange coupling 2J. Since for laccase 2J > -
The active site in Rhus vernicifera laccase contains four copper ions that together catalyze the four-electron reduction of oxygen to water with concomitant oxidation of substrate (1). Based on their EPR properties, these Cu(II) centers have been classified as type 1 or blue (All < 90 x 10-4 cm-1), type 2 or normal (All > 140 x 10-4 cm-'), and type 3 or coupled binuclear (nondetectable by EPR). Laccase, the simplest of the multicopper oxidases (2, 3), contains only one of each of these Cu(II) types and, hence, provides the most appropriate system for determining how dioxygen bonding and reactivity at the oxidase active site differs from that in the hemocyanins (4) and tyrosinase (5), which contain only a coupled binuclear copper site. In the latter proteins, dioxygen binds reversibly as peroxide and bridges the binuclear Cu(II) site in a u-1,2 coordination geometry (6). An important simplification for study of the type 3 site has been the preparation of a type 2 Cu(II)-depleted (T2D) laccase form (7). As in hemocyanin, the coupled binuclear Cu(II) site in fully oxidized (8) T2D laccase contains two tetragonal Cu(II) ions that are antiferromagnetically coupled by superexchange through an endogenous protein bridge (OR-). However, whereas exogenous anions bind equatorially and bridge the binuclear Cu(II) site in the hemocyanins
Abbreviations: MCD, magnetic circular dichroism; LTMCD, lowtemperature MCD; T2D, type 2 copper depleted; CT, charge trans-
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
fer. *To whom correspondence should be addressed.
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
3063
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Chemistry: Allendorf et aL
Proc. NatL Acad Sci USA 82 (1985)
550 cm-' (14, 15), the S=1 state is not thermally accessible. An MCD band associated with a CT transition from the S= 0 ground state will be temperature independent and weaker by a factor of 100-1000 at 10 K than bands associated with
8000
paramagnetic ground
6000
states
(16). Thus, through LTMCD
studies of laccase, one can determine whether the ground state of the Cu(II) associated with an N- -- Cu(II) CT transition is paramagnetic and hence due to type 2 Cu(II) or is diamagnetic and thus associated with the type 3 site. These LTMCD studies strongly suggest that exogenous anions directly coordinate to and bridge between the type 2 and type 3 Cu(II) centers, thereby defining a new type of Cu(II) active site, which is trinuclear.
4000
2000
MATERIALS AND METHODS
Laccase was purified (17, 18) from the acetone powder (Saito and Company, Osaka, Japan) of the Japanese lacquer tree, Rhus vernicifera. Routinely, the protein solutions were concentrated to =0.4 mM by using an Amicon YM-10 membrane. To obtain glasses of high optical quality, the protein was dialyzed into 50% (vol/vol) glycerol/0.1 M potassium phosphate, pH 6.0, which further concentrated the sample to =1 mM (the protein concentration for all spectra presented here). For a given titration experiment, this protein was divided into 300- to 400-1ul aliquots, and 5 A.l of N- solution (of appropriate N- concentration) was added directly to each sample. These solutions were then equilibrated for -16 hr at 40C. Absorption spectra were recorded by using two quartz disks spaced by a rubber gasket (1.4-mm path length) on a Cary 17 spectrophotometer. The sample was mounted on an Air Products LT-3-110 Heli-tran liquid helium cryotip, which was fixed in the Cary 17 sample compartment. A Lakeshore Cryotronics DTC-500 allowed spectra to be taken from 80300 K. Equilibrium binding constants were calculated as described (19). EPR spectra were recorded at 9.39 GHz on a Bruker ER 220 D-SRC spectrometer. An Air Products LTD-3-110 Helitran liquid helium transfer refrigerator and the Lakeshore temperature controller maintained samples at 8 K. MCD spectra were recorded on a JASCO J500C spectropolarimeter with a modified sample compartment to accommodate a superconducting magnet. Magnetic fields up to 60 kG were produced by an Oxford SM4 superconducting magnet/cryostat. Samples were mounted as described for the low-temperature absorption spectra. Depolarization was measured by recording the CD spectrum of a nickel tartrate solution placed before and after the sample (zero magnetic field) and was rarely observed. Sample temperatures were measured with a carbon glass resistor, calibrated from 1.5300 K, by Cryogenic Calibrations (Pitchcott, Aylesbury, Buckinghamshire, U.K.). The temperature was maintained (±0.05 K) by an Oxford DTC-2 temperature controller connected to a Rh/Fe resistance thermometer.
e
-0.50
.25x/c 300
400
500
600
700
Waveengt,0n Wavelength, nm FiG. 2. Native laccase spectra. The upper absorption spectra are at 298 K (-) and 77 K (---). The lower spectra are variable-temperature MCD spectra at 50 kG: a, 59.8 K; b, 20.4 K; c, 11.8 K; d, 4.75 K; and e, 1.75 K. Units of E are liter/mol-cmkG.
4.0 -.
2.0 -
0.30f 0.3
RESULTS AND DISCUSSION Fig. 2 shows the absorption at 298 and 77 K and the LTMCD spectra at 1.7-59.8 K of native laccase between 800 and 300 nm. As the temperature was decreased, the 614-nm region of the absorption spectrum sharpened, similar to that previously reported (20). In the LTMCD spectra of Fig. 2, the strong 1/T dependence of each of the bands (610, 55, 450, and 345 nm) is evident and indicates that they are associated with the paramagnetic types 1 and 2 Cu(II) sites. Upon N- titration of native laccase, several sets of overlapping spectral features appeared in the 550- to 300-nm spectral region, which are assigned as N- -+Cu(II) CT transitions (Fig. 3). Those features observed at N- concentra-
-0.60 Wavelength, nm FIG. 3. Titration of native laccase with N3 . (Upper) Absorption
spectra at 298 K. (Lower) LTMCD (4.9 K) spectra at 50 kG. Native laccase; ---, 0.25 and 0.50 protein equivalents of N3; 2.5 and 9.0 protein equivalents of N; ., 38.0 protein equivalents of N-.
Proc. NatL Acad Sci. USA 82 (1985)
Chemistry: Allendorf et aL tions s 0.5 protein equivalents (i.e., c 0.5 equivalents of N3 per equivalent of laccase) are designated "high-affinity" based on their high binding constant (K > 10 M' at 298 and 4.9 K). The second set appears at N- concentrations > 0.5 protein equivalents and is designated "low-affinity" binding (K = 102-103 M-1 at 298 and 4.9 K). The low-affinity features further display two behaviors: for N- concentrations up to 9.0 protein equivalents, the intensity of the LTMCD features increases continuously, following that of the absorption bands. However, at higher concentrations dramatic decreases occur in some of the LTMCD features, while the absorption bands continue to increase. Because the low-affinity features dominate the spectra, these spectral changes are examined first. Difference absorption and LTMCD spectra, relative to a baseline N- concentration of 0.5 protein equivalents (to correct for the high-affinity N- features; see above) are shown in Fig. 4 for a titration of native laccase with NW. In the 550to 370-nm region of the 298 K absorption spectrum, a strong peak is observed at =400 nm with a broad shoulder centered at =500 nm. In the LTMCD spectrum at 4.9 K (Fig. 4 Lower), three sharp, negative bands appear at 485, 442, and 340 nm with an equally intense positive feature at 385 nm. Since MCD intensity is proportional to absorption intensity, the changes observed in an MCD band with the addition of N3 should reflect the changes occurring in the N - *Cu(II) CT absorption feature corresponding to that MCD band. This Wavelength, nm 400 333
500
:C
1500/ b
A
1000
5000
3065
parallel behavior is observed in Fig. 4 for the 485-nm LTMCD and 500-nm absorption bands; both increase continuously as the concentration of N- is increased. Since there is a band in the LTMCD spectrum corresponding to the 500nm absorption feature, the ground state of this electronic transition must be paramagnetic. Therefore, these two features can be assigned to a single CT transition from N- to the type 2 Cu(II). The behavior of the remaining MCD bands in Fig. 4 is considerably more complex. Continuous intensity increases with increasing N- concentration are observed for the 400nm absorption band, but parallel changes in the MCD spectrum do not occur. Initially, simultaneous (as evidenced by the common crossover points at 351 and 422 nm) and continuous increases in the MCD intensity are seen at 340, 385, and 442 nm as the N- concentration increases from 0.5 to 9.0 protein equivalents. When the N- concentration is increased further, however, the intensity of all three bands decreases, preventing a simple correlation between the absorption band at 400 nm and the MCD features. In the EPR spectrum (Fig. 5), however, a new signal is observed between 3200 and 3800 G at 9 protein equivalents suggests that an additional N- binds, perhaps shifting the features to a different spectral region.
a
0.50-
PI
b
3.0
2.5
2.0
Energy, 10 4xcm1 FIG. 4. Low-affinity N- spectral features. (Upper) Difference spectra at 298 K relative to an absorption baseline with N- concentration of 0.5 protein equivalents. Baseline variations at 77 K because of stray light and cracking of the sample glass preclude the calculation of accurate difference spectra at this temperature. (Lower) LTMCD (4.9 K) difference spectra at N_ concentrations of 2.5 (spectrum a), 9.0 (spectrum b), and 38.0 (spectrum c) protein equivalents.
0
3000
6000
Gauss FIG. 5. EPR spectrum at 8 K of native laccase (trace a) and native laccase with 9 protein equivalents of N_ (trace b). Arrow points to g = 1.86.
Chemistry: Allendorf et aL
3066
Proc. NatL Acad Sci. USA 82 (1985)
2.0
1.5-
-~~
o
0.5
0
10
30 20 Protein Equivalents N3
40
FIG. 6. Changes of EPR, LTMCD, and absorption intensities with N_ concentration, normalized to the intensity of the spectrum with N_ at 9 protein equivalents for each type of spectroscopy. , LTMCD (4.9 K) at 485 nm; ---, A4w at 298 K; -.-, LTMCD (4.9 K) at 385 nm (intensity adjusted to maintain constant crossover points at 351 and 422 nm); . , g = 1.86 EPR signal at 8 K. All intensities were determined by using the baseline with N_ concentration at 0.5 protein equivalents. Error bars with double hatch marks correspond to the EPR signal. The graph represents the observed behavior of these spectral features in four sets of experiments.
Alternatively, the changes in the absorption band at =400 nm as a function of N- concentration do not reflect the intensity decrease in the LTMCD spectrum at 385 nm (Fig. 6). Thus, an additional CT spectral feature must be contributing to the absorption spectrum at 400 nm. The quantitative correlation of the LTMCD intensity changes with those of the type 3 EPR signal (Fig. 6) requires that within ± 5%, all of the 385-nm LTMCD signal be associated with the uncoupled type 3 sites. This indicates that the additional absorption feature has no measurable LTMCD intensity associated with it. From the N- binding constant calculated from the 77 K absorption at 400 nm combined with the integrated EPR intensity, it is estimated that .70% of the absorption intensity has no LTMCD intensity; hence, it must arise from a transition associated with a diamagnetic site. Therefore, this additional feature, which dominates the absorption spectrum at 400 nm, can be assigned to an N- -3 type 3 (coupled) Cu(II) CT transition. The intensity changes for the 485-nm LTMCD band asso-
ciated with the paramagnetic type 2 sites are also included in Fig. 6. This transition clearly shows a behavior with N3 concentration that is qualitatively similar to that of the 400-nm absorption band (i.e., both continuously increase), and binding constants calculated for the two bands are estimated to be the same within experimental error (K 102-103 M-1). Thus, low-affinity N- binding generates CT transitions from both a diamagnetic (400-nm absorption band) and a paramagnetic (385-nm LTMCD band) ground state with similar binding constants. This demonstrates that either a single N3 bridges the type 2 and coupled binuclear type 3 Cu(II) sites, or alternatively, that two N- molecules are binding with very similar binding constants at the laccase active site, one to the type 2 and one to the type 3 center. The assignment of both the 485-nm LTMCD and the 400nm absorption features to a single bridging N- is supported
by related studies at pH 7.0. Earlier work (11, 13) has shown that the 400-nm absorption intensity is greatly reduced at high pH due to a reduction of this N- binding constant; we find a similar decrease of the 485-nm LTMCD intensity. It is unlikely that binding of N- to a binuclear type 3 vs. a mononuclear type 2 Cu(II) site would exhibit this same pH effect. Additionally, the binding constant of high-affinity N-3 which corresponds to N- binding to an oxidized type 2 site in the presence of a reduced type 3 site (see below), is not significantly affected by this pH change. These results indicate that the oxidation state of the type 3 site strongly affects both the N- binding constant of the type 2 site (low vs. high affinity) and the pH perturbation of that binding. Finally, N3 binding studies of fluoride-treated laccase indicate that fluoride binding at the type 2 Cu(II) directly competes with N3 binding at the type 3 center (unpublished data). This reciprocal interaction between the type 2 and type 3 sites together with the correlated behavior of the 400-nm absorption and 485-nm LTMCD features under a variety of conditions strongly indicates that a single N- bridges the type 2 and type 3 sites. When low concentrations of N3 (
