Paramagnetic Resonance of Metallobiomolecules - American

Chapter 16

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Paramagnetic NMR of Electron Transfer Copper Proteins 1

2,*

Claudio Ο. Fernández and Alejandro J. Vila 1

LANAIS-RMN, CONICET, University of Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina Instituto de Biología Molecular y Celular de Rosario (IBR), University of Rosario, Suipacha 531, 2000 Rosario, Argentina 2

l

Paramagnetic H NMR studies of electron transfer copper sites in proteins have been possible due to recent progress in the field. This has enabled the analysis of the electron spin derealization onto the different ligands in blue copper and Cu sites. The weakly interacting axial ligands play an important role in tuning the electronic structure of both sites. A

Copper in living systems exists in two oxidation states: Cu(I) and Cu(II). This redox couple is adaptable to the biological redox potential range. Intra- and intermolecular electron transfer by copper centers is performed by the mononuclear blue (type 1) copper and binuclear C u sites (7). Type 1 centers are present in the so-called blue copper proteins (BCP's) found in bacteria and plants, as well as in multicopper enzymes, present in different organisms (2; J). The binuclear purple Cu center is the electron entry port of terminal oxidases, and it has not been found in single-domain soluble proteins (4). Cu(II) is a d , open shell transition metal ion, with one unpaired electron located in the d . orbital. Instead, Cu(I) possesses a d closed shell, being diamagnetic. Blue copper sites use the Cu(II)/Cu(I) redox pair. In the binuclear A

A

9

2

x

2

10

y

© 2003 American Chemical Society In Paramagnetic Resonance of Metallobiomolecules; Telser, Joshua; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

287

288 C u center, the two physiologically relevant redox states are the fully reduced (Cu(I),Cu(I)), and the oxidized, mixed-valence Cu(I),Cu(II). Therefore, the oxidized states of both blue and Cu sites are S=l/2. In these forms, the coupling between the unpaired electron and the nearby nuclear spins in the protein affects both the chemical shifts and the relaxation rates of these nuclei. Paramagnetic Cu(II) centers in proteins were considered for a long time as not amenable for NMR studies, since the electron relaxation rates of Cu(II) gave rise to extreme line broadening. In 1996, the first paramagnetic H NMR spectra of a blue copper center and of a Cu site in the oxidized forms were reported by Canters (Leiden) (5) and Bertini and Luchinat (Florence) groups (d), respectively. More recently, the full assignment of the paramagnetic spectrum of an oxidized blue copper site was pursued by the Florence lab (7), and the first solution structure of an oxidized blue copper protein was reported (8). These efforts gave breadth to several NMR studies, that we will review in this Chapter. A


A

l

A

The Electron-Nucleus Coupling The electron-nucleus hyperfine coupling occurs via two mechanisms: contact (through chemical bonds) and dipolar (through space). Both mechanisms affect the chemical shifts and nuclear relaxation rates. The observed chemical shifts in paramagnetic systems are the result of three contributions: 4bs

=

£dia + 4on + $pc

(1)

where is the chemical shift of the observed nucleus in an analogous diamagnetic system, δ^ is the Fermi contact shift due to the unpaired electron density on the nucleus, and δ^ is the pseudocontact shift (see below). The Fermi contact contribution is given by (9; 10): ζ

_A c m

h

g S(S + l) eMB

( 2 )

l kT Yl

where A is the hyperfine coupling constant and # is the nuclear gyromagnetic ratio. This contribution is operative through chemical bonds, and reflects electron derealization through the molecular orbitals. Contact shifts directly provide an estimate of the the spin density derealization and, indirectly, information on the metal-ligand orbital overlap. In blue copper proteins, contact shift values of protons from the copper ligands range from ~ 900-300 ppm for cysteine signals to less than 100 ppm for histidine resonances (7; 11; 12).

In Paramagnetic Resonance of Metallobiomolecules; Telser, Joshua; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

289 The electron-nucleus dipolar interaction gives rise to a pseudocontact contribution (5 ) to the shifts (JO). This term can be evaluated if the protein structure and the magnetic susceptibility anisotropy tensor are known. Due to the low magnetic anisotropy of Cu(II) centers, 5 can be calculated by neglecting the rhombic contribution (5; 7), according to: pc


pc

where r is the proton-copper distance, θ is the angle between the metal-proton vector and the magnetic ζ axis, and the g tensor parameters are defined by g| = gzz and gj_=(gxx + gyy)/2. In Cu(II) proteins, 8 values on metal ligand protons are expected to be smaller than 3.2 ppm (5;7;11;12). This value is negligible for the large shifts observed for β-Cys protons (we will consider ô = ô ) . The possibility of detecting H NMR signals in Cu(H) proteins depends on the nuclear relaxation rates, that depend on the electron relaxation rates (tj ). Copper(II) sites in proteins possess distinctive electron relaxation times which confer idiosyncratic spectral properties to each type of metal center (13). Catalytic mononuclear Cu(II) centers display slow electron relaxation rates (%' < 10 s"), thus giving rise to very broad NMR lines which escape direct detection. In blue copper sites, the unpaired electron relaxes faster ( τ / > 10 s"), due to the existence of low-lying electronic excited states arising from the trigonal nature of the metal center (14). Paramagnetic NMR spectra of blue copper proteins can then be recorded, even if the resonances are still broad, and the assignment is not straightforward (5;7). The oxidized form of C u is a mixed-valence S=l/2 system, that displays even faster electron relaxation rates (> lO ^" ) (15), thus giving rise to sharp lines, which can be assigned through NOE and 2D experiments (6). (

pc

c o n

obs

l

1

x

s

9

1

1

9

1

A

1

1

Mononuclear Blue Copper Sites Blue copper sites in the oxidized form are characterized by unusual spectroscopic features such as an intense charge transfer band in the absorption spectrum at 600 nm, and a small hyperfine coupling constant in the EPR spectrum (1;16). All blue sites present a single copper ion coordinated to a Cys and two His residues, located approximately in the same plane (Figure 1) (2). Despite these conserved features, the electronic structure of blue sites is finely tuned by small perturbations that give rise to different spectroscopic fingerprints (16; 17). Blue copper sites have been classified into "classic" and perturbed sites by Solomon and coworkers, based on their spectroscopic properties (16;18).



290

Figure L Schematic drawing of the blue copper sites in: (A) plastocyanin, (B) pseudoazurin, (C) stellacyanin and (D) azurin. Classic blue copper sites (present in plastocyanin, amicyanin and azurin) exhibit an intense absorption feature at -600 nm, a weak band at -450 nm, and nearly axial EPR spectra. These centers are characterized by a weak bonding interaction with an axial methionine ligand (Figure 1A) (19;20). In azurin, a backbone oxygen from a Gly residue is located on the opposite side of the equatorial plane, at - 3 Â from the copper ion (Figure ID). As a consequence, the copper ion is located almost coplanar to the N i(His)-N i(His)-S (Cys) plane. Perturbed centers can be distinguished by an increased absorption ratio of the 450 nm and 600 nm bands and markedly rhombic EPR spectra (17). These perturbations occur when stronger copper-axial ligand interactions are present. Perturbed centers can arisefroma tetragonal distortion with an axial Met ligand in a different orientation, as in pseudoazurin (Figure IB), the cucumber basic protein and rusticyanin,(27-25) orfroma tetrahedral distortion, when a Gin ligand replaces the Met, as in stellacyanin (Figure 1C) (24;25). 5

S

y

*H N M R Spectra and Signal Assignment Strategy Assignment of paramagnetic resonances in blue Cu(II) sites cannot be performed by NOE or 2D techniques, due to the large line widths. Instead, they may be assigned by saturation transfer experiments in a sample containing a mixture of the oxidized and reduced forms of the protein. Irradiation of signals of the Cu(II) species gives rise to a saturation transfer to the corresponding resonances of the same nuclei in the diamagnetic Cu(I) state (5;7;26). These



291

C-His Figure 2. *HNMR spectra of Cu(II) plastocyanin (Pc), azurin (Az), stellacyanin (St), pseudoazurin (Pz) and rusticyanin (Rc) (7;11;12). The groups of signals labeled with Ν and C correspond to the N-and C-tenninal His ligands, respectively. Resonances from the Met and Asn residues are indicated. resonances can be assigned by standard NMR protocols, and the assignments can be transferred to the paramagnetic species. This strategy has led to the assignment of resonances corresponding to the His and the axial ligands in different blue copper sites (Figure 2) (5;8;11;12;26;27).


292 The β-ΟΗ Cys signals bound to Cu(II) cannot be directly detected due to their large line widths. However, they can be assigned through "blind" saturation transfer, a strategy devised by Bertini, Luchinat and coworkers.(7) In this experiment, selective saturation is applied over a large spectral range (± 2000 ppm) by shifting the position of the decoupler, even if no signals are observed. If a signal broadened beyond detectable limits is present in the irradiated region, a saturation transfer will be observed. This effect will be maximum when the decoupler exactly matches thefrequencyof the broad (unobserved) signal, and will fade out when irradiation is off-resonance. The Cys signals can be reconstructed by plotting the saturation transfer response vs. the irradiation frequency. This strategy has been recently applied to record, assign and analyze the paramagnetic H NMR spectra of Cu(II) plastocyanin, azurin (two classic blue sites), stellacyanin (a tetrahedrally distorted site), rusticyanin and pseudoazurin (two tetragonally distorted sites). These studies have allowed us and others to map the electron derealization on *H nuclei from the different metal ligands in blue copper proteins (8;11;12).


2

l

Metal-ligand interactions The Cu-His Interaction The chemical shifts for each type of proton span rather narrow shift ranges for both His resonances (Figure 2). This suggests that: (1) the unpaired spin density in both histidine ligands is fairly constant, either in classic or distorted sites; and (2) a similar electron derealization pattern is operative in the imidazole groups (12). These conclusions are feasible provided the orientation of the copper orbitals in the xy plane with respect to the histidine ligands is similar in all blue sites. This concurs with the proposal from Van Gastel et al., who pointed out that the magnetic ζ axis in different BCP's is nearly perpendicular to the plane defined by the copper ion and the coordinated N (His)(2S). 61

The Cu-Cys Interaction The p-CH Cys protons display large hyperfine coupling constants, consistent with a high Cu(II)-Cys covalence (Figure 3). In addition, these signals display drastically different chemical shifts among the different proteins (from 240 to 850 ppm, that correspond to hyperfine coupling constants from 8 to 28 MHz). These variations reveal changes in the Cu(H)-Cys covalence of the different sites. 2


293 The observed β-ΟΗ Cys shifts depend on the Cu-S-C -H dihedral angles. Instead, the average chemical shift of these nuclei (δ ) does not reflect a strong dependence on the Cys conformation.(/2;29) The β-ΟΗ Cys Sy values follow this trend: Cu(II)-azurin (825 ppm) > Cu(II)-plastocyanin (570 ppm) > Cu(II)pseudoazurin (450 ppm) > Cu(II)-stellacyanin (415 ppm) > Cu(II)-rusticyanin (270 ppm) (Figure 3). Pseudoazurin and rusticyanin exhibit almost identical UV-vis and EPR spectra,(30;5i) but quite different Cu(II)-Cys covalencies. These differences in the NMR spectrum parallel results from X-ray data (the Cu-S (Cys) distance in rusticyanin is 0.13 Â longer than in pseudoazurin).(22;25) A plot of Sy vs. r s for the studied proteins shows a linear trend, except for azurin (Figure 4A). In blue copper sites, the unpaired electron is mostly confined to the copper d . orbital. Then, at afixedr s, the copper-sulfur overlap is expected to depend on the position of the sulfur atom respect to the xy plane. If 5y is plotted towards a corrected r s parameter, in which the copper-sulfur distance is weighed by the cos of the angle between the Cu-S bond and the xy plane, a linear trend is found for all blue sites (Figure 4B). The fact that the P-CH Cys Sy values can be accounted for by geometric features of the metal site that determine the coppersulfiir overlap further validates the use of this parameter as a reliable estimate of the electron spin density on the S (Cys) atom. 2

p

p

υ2


2

2

Y

2

Cu

2

x

2

y

Cu

2

Cu

2

2

y

The Cu-Met Interaction The axial ligand is weakly bound in blue copper proteins, and thus axial ligand resonances display small or null electron spin density. In azurin, no hyperfine-shifted resonances corresponding to the axial Met or Gly ligands could be detected. This correlates with the long Cu-S (Met) bond in azurin (3.0 Â). Instead, hyperfine contributions to the Met resonances have been identified in plastocyanin, amicyanin, pseudoazurin and rusticyanin. In Cu(II)-plastocyanin (8) and Cu(II)-amicyanin,(5) the y-CH Met resonances show net electron spin density. Instead, hyperfine shifts in the 8-CH Met moieties have been identified in Cu(II)-pseudoazurin and rusticyanin.(72) The different electron derealization patterns can be attributed to the fact that the Met side chain adopts a trans conformation in classic blue sites (plastocyanin and amicyanin), and a gauche conformation in tetragonally distorted sites (pseudoazurin and rusticyanin). In all cases, the larger hyperfine shifts correlate with shorter Cu-S t bonds. 5

2

3

Me

Electron delocalization through hydrogen bonds In most blue copper proteins, the amide NH proton of an Asn residue forms a hydrogen bond with the S (Cys) atom (2). A net electron spin density has been found on the Asn NH nucleus in four different blue copper proteins, that reveals Y



δ (ppm)

2500 2000 1500 1000 500

Azurin

δ (ppm)

2500 2000 1500 1000 500 0

δ (ppm)

2500 2000 1500 1000 500

Λ \A

Pseudoazurin

0

ο (ppm)

2500 2000 1500 1000 500

Stellacyanin

0

Rusticyanin

δ (ppm)

0

Λ 2500 2000 1500 1000 500

Figure 3. Reconstruction of the β-Cys proton resonances from blind irradiation profiles offiveblue copper proteins.

0

Plastocyanin


V©


200

2.05

300 -

400 -

500 -

2.10

•St

2.25

• Rc 2.20 2.30

2

200

300

400

2.1

2.2

« Azu

2.3

psAz»

• PC

2.4

St

Figure 4. Plot of the β-Cys 5y parameter vs. (A) the experimental coppersulfur distance, and (B) the copper-sulfur distance corrected for the Cys deviation from the equatorial ligand plane.

2.15

»psAz

500

600

• Pc

600

800 700

> Azu

m

*i«(PP > 900

700

800

*ii2(PPm) 900


2.5

• Rc

Β

2.6

in

SO

296 that electron derealization is operative through this hydrogen bond. The contact shift of the Asn NH signal shows a linear correlation with the electron spin density on the Sy(Cys) (quantified by the p-CH Cys δ value) in all studied blue sites, except in rusticyanin (Figure 5) (12). Rusticyanin lacks the conserved Asn, that is replaced by a Ser. This results in a higher unpaired spin density in the Ser NH proton compared to that expected according to the trend shown in Figure 5. This suggests that the replacement of an Asn by a Ser gives rise to a perturbed hydrogen bond interaction. It has been suggested that this perturbation could contribute to the unusually high redox potential (680 mV) of rusticyanin (12).


2

υ ι

MetaUligand interplay in blue copper sites A comparative analysis of the different copper sites reveals a subtle interplay between the different copper-ligand interactions. In azurin, the Cu(II) ion lies in the His Cys plane, and the axial ligands (Metl21 and Gly45) are located at ~ 3Âfromthe metal ion at both sides of the equatorial ligand plane (Figure IB) (20). This coplanarity gives rise to the largest Cu(II)-Cys covalence among blue sites, with no electron spin density in the axial ligands. In 2

-20

St* psAz

-24

-28 ô NH(ppm)

PC·

Rc*

con

-32

-36 Λ

-40 200

300

400

500

600

800

700

900

h (ppm) m

Figure 5. Plot of the NH amide contact shift vs. the 8

m

β-Cys parameter.



297 plastocyanin and amicyanin, with one axial ligand (a Met), the copper moves closer to the Met ligand (79/52), resulting in a net electron spin density in the Met side chain and reducing the Cu(II)-Cys overlap. In stellacyanin, the axial bond between copper and the Gin oxygen imposes a tetrahedral distortion to the site, which further weakens the Cu(H)-Cys bond (24;25). These results reveal that in classic and tetrahedrally distorted blue sites, a stronger interaction with the axial ligand results in a weakened electron derealization in the Cu(II)-Cys bond (77). The electron spin density in the Cys ligands in tetragonally distorted blue sites (pseudoazurin and rusticyanin) is decreased compared to classic sites (azurin and plastocyanin), but this is not related to a stronger interaction with the axial ligand. Instead, the Cu-Cys and the Cu-Met interaction are independently tuned by the protein folding around the metal site in tetragonally distorted blue copper sites (72).

Electronic structure of C U A sites C u is a binuclear center, where the copper ions are bridged by the two sulfur atoms of Cys ligands, forming an almost planar Cu S rhombic structure with a metal-to-metal distance ranging between 2.3-2.6 Â (33-36). One of the copper ions is coordinated also to N i(His) and a S§(Met) donor atoms, whereas the other copper ion is coordinated by a N i(His) and a backbone carbonyl (Figure 6). These geometrical features are conserved in all structurally characterized Cu centers (33-36). The weak Met and carbonyl ligands may help to maintain the site architecture and to regulate its properties (37). C u centers exist in two redox states: [Cu(II),Cu(I)] and [Cu(I),Cu(I)]. The oxidized species is a fully delocalized type III mixed-valence pair (formally two Cu +1.5 ions), as early revealed by EPR spectroscopy (38). These systems display sharper NMR lines than BCP's due to a shorter electron relaxation time A

2

2

S

S

A

A

Figure 6. Schematic drawing ofT.thermophilus Cu site) (36). A


298 11

of the paramagnetic center (« 10" s), giving rise to well-resolved paramagnetic *H NMR spectra (Figure 7) (6). NMR studies are available for the native C u centers from the solublefragmentsof the T. thermophilus (6;39), P. denitrificans (40), P. versutus (41), and B.subtilis oxidases (42), P. stutzeri N 0 reductase (43) as well as for engineered C u sites in amicyanin (44)md E. coli quinol oxidase (45). The proton signals of each His ligand, which are found up to 40 ppm (Figure 7), can be assigned by ID NOE or NOES Y experiments (6). They can be assigned to specific residues based on the fact that only one of them is solvent exposed, as it also occurs in BCP's. The two His residues show different amounts of delocalized spin density in each system. These differences might reflect different tiltings of the imidazoles with respect to the Cu S rhombus, likely being modulated by the Cu-axial ligand distances (37). The four (î-CH Cys resonances are shifted well downfield, spanning a broad range of chemical shifts from 50 to 450 ppm (Figure 7). Three are very broad, and usually are found between 200 and 450 ppm whereas a fourth, sharper one, falls in the range 50-110 ppm. In some cases, line broadening is so drastic that the broader resonances cannot be detected. This problem can be overcome by selective deuteration of the β - Ο Ι Cys protons, and collection of the H NMR spectrum (40). The assignment of the P-CH Cys signals has been performed from the analysis of the Cu-S^-C^H^ dihedral angles and on the basis of the shifts temperature dependence. The Cys resonances exhibit strong deviations from Curie behavior, that can be explained by assuming partial population of low-energy excited states (41;46). Analysis of the Cys signal pattern has confirmed that the ground level in C u sites is a a * orbital, and the excited state is a n level. The a */n energy gap can be estimated by analysis of the temperature dependence of the Cys chemical shifts (41).

A

2


A

2

2

2

2

2

2

A

u

u

u

u

No signals from any of the axial ligands are observed significantly shifted outside of the diamagnetic envelope. This is indicative of a very weak interaction of both Met and Glu ligands with the copper ions and a small or negligible amount of spin density delocalized onto them. The Metl60Gln mutant in the C u soluble fragment from Thermus thermophilus provided the first stable, mixed valence axial ligand mutant in a soluble C u fragment(39;47). A *H NMR study of this mutant reveals no net electron spin density in the axial ligands, and that the Cu-Cys covalence is unaltered (39). These results indicate that no interplay between axial ligands and the Cu-Cys bonds is present in C u sites, in contrast with blue copper proteins. Instead, other changes were observed. The σ„*/π energy gap is increased with respect to the WT protein. This picture is consistent with changes in the electron relaxation time in the mutant. Therefore, the energy of the low-lying excited state can be tuned by the interaction of the copper atoms with the axial ligands (39). A

A

A

α



299

,

,

140

1

ι

140

,

,

120

1 120

,

,

100

1

1 100

,

,

80 δ (ppm)

1

1

,

,

60

1

1

80 δ (ppm)

60

,

,

40

I

I

1 20

I

40

]

Γ" 20

Figure 7. Downfield region of the HNMR spectra of wild type and MetloOGln mutant of the Cu ~Thermus fragment (6, 39) A


300 The two His ligands are less alike in MetloOGln than in the WT protein due to the selective perturbation of Hisll4, whereas Hisl57 resonances remain unaltered. His 114 is bound to the same copper atom as the engineered Glnl60 residue (Figure 6). The engineered Gin ligand in the mutant affects the copperHis interaction, either by tilting His 114 relative to its orientation in the WT protein, or by strengthening the Cu-His bond. These results provide experimental evidence that each of the two halves of the C u core can be modulated independently. Axial ligand perturbations of the Cui or Cu coordination environment thus may provide unique regulatory possibilities of the C u reactivity in the full oxidase. Comparison of Cu features with blue copper sites sheds some light on the role of axial ligands in these related systems. Stronger copper-axial ligand interactions in blue copper sites (such as a Met-»Gln mutation) induce changes in the relative intensities of the charge transfer bands (16;17), give rise to rhombic EPR spectra and affect the Cu(II)-Cys covalence (16). In contrast, the charge transfer band pattern in Metl60Gln-Cu is only slightly altered with respect to that of the WT protein (47), the EPR spectrum is axial, and the CuCys covalence is not affected (39). It can be concluded that axial ligands in C u sites can exert perturbations in the Cu S rhombus that tune the energy of the n excited state, and determine the orientation of the His ligands. Both mechanisms could play a functional role in terminal oxidases by regulating intra- and intermolecular electron transfer.


A

2

A

A

A

A

2

2

u

Acknowledgments

Alejandro J.Vila thanks the Fundacion Antorchas and the Fogarty International Center- NIH (R03-TW000985-01) for supporting studies on copper proteins.

References

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