Plastocyanin copper site

The EPR spectrum of the blue copper protein plastocyanin (Figure 3C) has gu > g > 2.00, and thus the copper site must have a dx2 y2 ground state. First, we are interested in determining the orientation of the dx2 y2 orbital relative to the distorted tetrahedral geometry observed in the protein crystal structure. Single crystal EPR spectroscopy allowed us to obtain this orientation and located the unique (i.e., z) direction in this distorted site (29). Plastocyanin crystallizes in an orthorhombic space group with four symmetry related molecules in the unit cell. The orientation of the plastocyanin copper sites in the unit cell are shown in... 

Fig. 3. (a) Copper site in plastocyanin. (b) Ribbon drawing of the plastocyanin backbone, (c and d) Schematic of plastocyanin topology. 

It is noteworthy that the proximity of the copper sites in ceruloplasmin, and, indeed, the involvement of most of the correct ligand histidines, were predicted some time ago by Ryden (1982, 1984) strictly on the basis of sequence homologies to plastocyanin. A similar prediction was made for laccase based on sequence similarities around the cysteine regions (Briving et al, 1980). Proximity of the type II site to the type III site (e.g., a trinuclear site) was also predicted by Solomon and co-workers (Allen-dorf et al., 1985 Spira-Solomon et al, 1986) on the basis of spectroscopic analysis of azide binding to laccase. What could not have been foreseen... 

Blue copper proteins, 36 323, 377-378, see also Azurin Plastocyanin active site protonations, 36 396-398 charge, 36 398-401 classification, 36 378-379 comparison with rubredoxin, 36 404 coordinated amino acid spacing, 36 399 cucumber basic protein, 36 390 electron transfer routes, 36 403-404 electron transport, 36 378 EXAFS studies, 36 390-391 functional role, 36 382-383 occurrence, 36 379-382 properties, 36 380 pseudoazurin, 36 389-390 reduction potentials, 36 393-396 self-exchange rate constants, 36 401-403 UV-VIS spectra, 36 391-393 Blue species... 

Fig. 7 Geometry of a typical type I copper site in plastocyanin. Bond lengths and bond angles are relatively constant upon reduction or oxidation of the copper site (J. M. Guss, P. R. Harrowell, M. Murata et al.,J. Mol. Biol. 1986, 792, 361-387).

Having elucidated, in combination with X-ray structural data, the characteristics of the copper site coordination in blue proteins in extenso, the challenge for EPR spectroscopy (and other techniques) is now to find ways to model the electron transfer (ET) in a realistic fashion. At present EPR is, however, mostly used to ascertain that the coordination of copper in the experimental ET chain models employed is not disturbed prior to ET. Plastocyanin is the electron carrier in photosynthesis. Indications of structural origins of impaired ET in... 

Pinacolone, o-(diphenylphosphino)benzoyl-coordination chemistry, 401 Piperidine, IV-hydroxy-metal complexes, 797 pA a values azole ligands, 77 Plant roots amino acids, 962 carboxylic acids, 962 Plastocyanin copper binding site, 557 copper(II) complexes, 772 copper(II) site in, 770 Platinum, dichlorobis(benzonitrile)-IR spectrum, 264 Platinum, cis-dichlorodianunine-antitumor activity, 34, 979 Platinum, ethylenebis(triphenylphosphine)-reactions with 5,6-dimethyl-2,l,3-benzothiadiazole, 194 Platinum blue formation, 265 Platinum complexes acetylacetone reactions, 380 amides, 491 amidines... 

Figure 3. The blue copper site in plastocyanin as determined by X-ray crystallography. Ligands (and copper-ligand bond lengths) are histidine 37 (2.OU A), cysteine 84 (2.13 A), histidine 87 (2.10 A) and methionine 92 ( 2.90 A). Reproduced with permission from Ref. 9. Copyright 1983, Journal of Molecular Biology.

Figure 13. Left ligand field energy-level diagram calculated for plastocyanin. Center contains energies and wavefunctions of the copper site. Energy levels determined after removing the rhombic distortions to give and C symmetries are shown in the left and right columns, respectively (from Ref. 11). Right electronic structural representation of the plastocyanin active site derived from ligand field calculations (from Ref. 11).

Extension of these edge studies (21) to the blue copper site in plastocyanin indicates that there is less than 1% p orbital mixing into d 2 2 and in particular that this involves the p and p orbitals. The Is to 3d 2 2 transition at 8979 eV occurs only wheX the polarization vector of the synchrotron radiation is in the xy plane. This can be seen from the Is to 3d transition reproduced in Figure 15 which appears only with the electric vector perpendicular to the Cu-methionine bond. As emphasized above, this p, p mixing cannot account for the small copper hyperfine coupling constant of... 

FIGURE 1.23 L-edge XAS spectra of the blue copper site in plastocyanin and D4h CuCI42, 53 Inset shows the relevant MO diagram and half-occupied HOMO wavefunction. 

FIGURE 1.25 Sulfur K-edge spectra for blue copper site in plastocyanin and the Cu(II) model complex tet b.49 Inset shows the MO diagram for a transition to the half-occupied HOMO. 

Fig. 5-9. The copper site in poplar plastocyanin based on the structure determined by Guss and Freeman (1983). Some of the bond angles and all of the copper-ligand bond distances are indicated.

The crystal structure of cucumber basic blue protein has now been refined to 3.0 A resolution (Adman, 1985). The protein consists of eight strands, only five of which form a P-sandwich and the protein has less P-sheet character than plastocyanin or azurin. The ligands to copper are provided by the side chains of His-39, Cys-79, His-84 and Met-89. The copper site has the N2SS coordination seen in plastocyanin. The imidazole rings of the His-39 and His-94 residues are exposed to the solvent providing a likely entry site for electon transfer to the copper centre. 

The additional effects in the aromatic region of the difference spectrum (250-300 nm) are probably caused by aromatic transitions which are influenced by the redox state of the copper. The shoulder at 270 nm, which occurs in all three proteins, could result from an increase in tyrosine absorption. In this context, it is interesting to recall that Tyr 108 (azurin numbering), which is relatively close to the proposed copper ligands Cys 112 and Met 121, is completely invariant both in azurin and plastocyanin and may therefore be an obligatory constituent of the copper site. 

Figure 4. Single-crystal EPR of poplar plastocyanin (29) orientation of the dxz.y2 orbital. A Unit cell and molecular orientation with respect to the applied magnetic field. B EPR spectra and simulations for the crystal orientations shown. C Orientation of the g direction and the dx2.yz orbital superimposed on the blue copper site.

Figure 8. Proposed electron transfer pathway in blue copper proteins. The plastocyanin wave function contours have been superimposed on the blue copper (type 1) site in ascorbate oxidase (40). The contour shows the substantial electron delocalization onto the cysteine Spir orbital that activates electron transfer to the trinuclear copper cluster at 12.5 A from the blue copper site. This low-energy, intense Cys Sp - Cu charge-transfer transition provides an effective hole superexchange mechanism for rapid long-range electron transfer between these sites (2, 3, 28).

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