Reconstitution and characterization of a divergent plastocyanin from the photosynthetic prokaryote, Prochlorothrix hollandica, expressed in Escherichia coli.
Plastocyanin (PC) is a copper protein that serves as a mobile electron carrier between cytochrome f and Photosystem I in the light reactions of photosynthesis. Despite large variability in amino acid sequences and isoelectric points, PCs from cyanobacterial and chloroplast sources reveal considerable similarities with respect to their secondary and tertiary structures. In this paper, we have expressed in Escherichia coli a PC from the prokaryote Prochlorothrix hollandica, and efficiently reconstituted the protein with copper under conditions yielding the characteristics of a native holoPC, as judged by redox titration (Eo' = +376 mV), near and far UV circular dichroism, and electron paramagnetic resonance (EPR) spectroscopy. By comparison of amino acid sequences, P. hollandica PC is the most divergent homolog identified to date, and analysis of this reconstituted preparation may reveal new insights as to the structural requirements for electron transport between the PC copper center and neighboring reaction partners.
Babu
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Center for Photochemical Sciences, Department of Biological Sciences, Bowling Green State University, Ohio 43403-0212, USA.
Arudchandran
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Hille
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Gross
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Bullerjahn
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GS
D002942:Circular Dichroism; D003001:Cloning, Molecular; D003300:Copper / Q000032:analysis; D000458:Cyanobacteria / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D010788:Photosynthesis; D010970:Plastocyanin / Q000096:biosynthesis* / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D003300:Copper; D010970:Plastocyanin