Brian F. Volkman, Ph.D.

The solution structure of the plastocyanin from from the photosynthetic prokaryote Prochlorothrix hollandica has been determined by homonuclear 1H NMR spectroscopy and torsion angle dynamics, in a collaboration with Dr. Charles Babu and Dr. George S. Bullerjahn (Bowling Green State University). A family of 19 structures was calculated from 1222 non-trivial distance constraints, yielding an ensemble backbone RMSD of 0.42 +/- 0.08 A for the backbone atoms. Despite low sequence similarity to other plastocyanins, the P. hollandica structure retains the same two-sheet beta-barrel tertiary structure.

NMR Structure of the plastocyanin from Prochlorothrix hollandica.

Plastocyanin (PC) is a small (~10 kDa), Type 1 copper protein that functions as an electron donor to Photosystem I (PSI) from cytochrome (cyt) f in both chloroplast systems and in some strains of cyanobacteria. In some lower eukaryotes and most cyanobacteria, a small c-type cytochrome (cyt) serves this function, and many of these organisms can synthesize both components, replacing PC with cyt c6 under conditions of copper limitation. PCs from cyanobacterial and chloroplast sources can be classified into four groups, I-IV, depending on their origin and primary structure. Whereas the chloroplast homologues exhibit a great deal of similarity at the level of primary structure, the cyanobacterial PCs are highly variable with respect to sequence and isoelectric point. Due to such variability, only 18 amino acids are universally conserved across this family of proteins, yet all retain a common tertiary structure and coordinate a single Cu ion near a hydrophobic region at the "top" of the molecule. The binding site is defined by a cysteine, a methionine and two histidine residues that form a distorted tetrahedral cage. His-87 (using the numbering from the poplar homologue) is a surface-exposed residue surrounded by the hydrophobic patch; it is likely that this residue yields the electron transport pathway into the PSI acceptor. In sum, PCs are b-sheet polypeptides that act as electron carriers having a midpoint redox potential of approximately +350 mV.

Two major structural features of PCs are the presence of two regions thought to be involved in binding the reaction partners: the hydrophobic patch on the top of the molecule in the vicinity of the H87 Cu ligand, and a negative patch composed of acidic amino acids common to chloroplast PCs, but absent in the cyanobacterial homologues. Whereas PCs in general all have a hydrophobic region, P. hollandica PC has a hydrophobic patch that is uniquely and substantially different at the level of amino acid sequence. The amino acid residues involved in forming the hydrophobic patch are G10, L12, P36, G89, and P86 (numbering based on the poplar sequence); indeed, G10 and L12 have been targets for site-directed mutagenesis in examining the role of this region in interactions with cyt f and PSI [10, 11]. Since these two residues were initially believed to be invariant in all PCs, we were quite surprised to find that the P. hollandica PC has a tyrosine and a proline residue at these respective positions. Determination of the solution structure of P. hollandica PC, and comparison to other known structures may help reveal the minimum structural features required for reaction partner binding and productive electron transfer.