Carbonic anhydrase activity of the photosystem II OEC33 protein from pea.
The purpose of this study was to identify the location of one of the two sources of carbonic anhydrase (CA) activity associated with the PSII complex in chloroplast membranes. We tested the hypothesis that the extrinsic 33 kDa protein, OEC33, associated with the oxygen-evolving complex (OEC), is one source of CA activity. We found that precursor OEC33 expressed in Escherichia coli exhibits CA activity, but the expressed precursors of OEC24 or OEC17 do not. The CA activity of OEC33 remained after treatment at 90 degrees C for 15 min. Additional biochemical evidence supports the hypothesis. Only those wash treatments that remove the OEC33 from PSII also remove CA activity. Both immunoblot and CA activity show that the CA tracks the OEC33, in parallel, when PSII undergoes washing at different CaCl2 concentrations. The OEC33 protein purified by HiTrap Q anion exchange chromatography has CA activity that is inhibited by an antibody against OEC33. PSII membranes washed with 1 M CaCl2 to remove OEC33 can be reconstituted either with extracted, purified, OEC33 or with the E. coli-expressed precursor OEC33. Reconstitution partially restores both oxygen evolution and CA activity. For maximal CA activity, OEC33 requires manganese as a cofactor.
Lu
Yih-Kuang
YK
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA.
Theg
Steven M
SM
Stemler
Alan J
AJ
D000906:Antibodies / Q000276:immunology; D002122:Calcium Chloride / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D002736:Chloroplasts / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D006358:Hot Temperature; D010100:Oxygen / Q000378:metabolism; D018532:Pisum sativum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D045332:Photosystem II Protein Complex / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D000906:Antibodies; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D011994:Recombinant Proteins; D002256:Carbonic Anhydrases; D002122:Calcium Chloride; D010100:Oxygen