Characterization of the metal binding environment of catalytic site 1 of chloroplast F1-ATPase from Chlamydomonas.
Metal ligands of the VO(2+)-adenosine diphosphate (ADP) complex bound to high-affinity catalytic site 1 of chloroplast F(1) adenosine triphosphatase (CF(1) ATPase) were characterized by electron paramagnetic resonance (EPR) spectroscopy. This EPR spectrum contains two EPR species designated E and F not observed when VO(2+)-nucleotide is bound to site 3 of CF(1). Site-directed mutations betaE197C, betaE197D, and betaE197S in Chlamydomonas CF(1) impair ATP synthase and ATPase activity catalyzed by CF(1)F(o) and soluble CF(1), respectively, indicating that this residue is important for enzyme function. These mutations caused large changes in the (51)V hyperfine tensors of VO(2+)-nucleotide bound to site 1 but not to site 3. Mutations to the Walker homology B aspartate betaD262C, betaD262H, and betaD262T of Chlamydomonas CF(1) caused similar effects on the EPR spectrum of VO(2+)-ADP bound to site 1. These results indicate that the conversion of the low-affinity site 3 conformation to high-affinity site 1 involves the incorporation betaE197 and betaD262 as metal ligands.
Chen
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The Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe 85287-1601, USA.
Hu
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Crampton
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Frasch
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D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D018698:Glutamic Acid / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013113:Spin Labels; D014638:Vanadates / Q000737:chemistry* / Q000378:metabolism*
D013113:Spin Labels; D001224:Aspartic Acid; D018698:Glutamic Acid; D014638:Vanadates; D000244:Adenosine Diphosphate; D006180:Proton-Translocating ATPases