TY - JOUR
T1 - pH-induced conformational changes in spinach ferredoxin
T2 - Steady-state and time-resolved fluorescence studies
AU - Kieleczawa, Jan
AU - France, Louisa L.
AU - Sutherland, John C.
AU - Hind, Geoffrey
N1 - Funding Information:
We thank Erwin London (State University of New York at Stony Brook) for the use of a Spex 212 Fluorolog spectrofluorimeter. The National Synchrotron Light Source at Brookhaven National Laboratory is supported by the Office of Chemical Research and the Office of Materials Research, USDOE, and the fluorometer at its port U9B is supported by the Office of Health and Environmental Research, USDOE.
Funding Information:
‘This work was supported by the Division of Energy Biosciences, Office of Basic Energy Sciences, United States Department of Energy (USDOE), Grant 89-37262-4544 from the Competitive Research Grants Office of the United States Department of Agriculture to G.H., and Grant GM34662 from the National Institutes of Health to J.C.S. ’ Present address: Plum Island Animal Disease Center, Greenport, NY 11944. ’ To whom correspondence should be addressed. 4 Abbreviations used: Fd, ferredoxin; DOPC, 1,2-dioleoyl-sn-glycero-
PY - 1992/10
Y1 - 1992/10
N2 - Steady-state and time-resolved fluorescence techniques were used to monitor pH-induced conformational changes in spinach ferredoxin. An increase was seen in the wave-length maximum of tryptophan-73 (Trp-73) emission, from 325 nm below pH 6.0 to 342 nm above pH 7.0, indicating significantly diminished hydrophobicity, at pH 7.0, in the environment of the indole ring. Raising the solution pH from 6.0 to 7.6 also decreased the binding of the detergent Brij-96, showing that the ferredoxin molecule as a whole became more hydrophilic at higher pH. Nonionic (acrylamide) and ionic (I- and Cs+) quenchers were used to probe the tryptophan environment. Trp-73 is partially shielded from I-, presumably by negatively charged residues, as predicted from the amino acid sequence and three-dimensional structure of plant-type ferredoxins. Ionic strength and pH effects on tryptophan fluorescence lifetimes follow a pattern common to single-tryptophan proteins: the emission decays can be fit to a biexponential model in which the lifetime of the excited state increases with increasing pH. The indication of a pH-induced conformational change in the range pH 6.0 to 7.6 is discussed with reference to the physiological association of ferredoxin with ferredoxin:NADP+ oxidoreductase and the rise in chloroplast stromal pH in the light.
AB - Steady-state and time-resolved fluorescence techniques were used to monitor pH-induced conformational changes in spinach ferredoxin. An increase was seen in the wave-length maximum of tryptophan-73 (Trp-73) emission, from 325 nm below pH 6.0 to 342 nm above pH 7.0, indicating significantly diminished hydrophobicity, at pH 7.0, in the environment of the indole ring. Raising the solution pH from 6.0 to 7.6 also decreased the binding of the detergent Brij-96, showing that the ferredoxin molecule as a whole became more hydrophilic at higher pH. Nonionic (acrylamide) and ionic (I- and Cs+) quenchers were used to probe the tryptophan environment. Trp-73 is partially shielded from I-, presumably by negatively charged residues, as predicted from the amino acid sequence and three-dimensional structure of plant-type ferredoxins. Ionic strength and pH effects on tryptophan fluorescence lifetimes follow a pattern common to single-tryptophan proteins: the emission decays can be fit to a biexponential model in which the lifetime of the excited state increases with increasing pH. The indication of a pH-induced conformational change in the range pH 6.0 to 7.6 is discussed with reference to the physiological association of ferredoxin with ferredoxin:NADP+ oxidoreductase and the rise in chloroplast stromal pH in the light.
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U2 - 10.1016/0003-9861(92)90094-D
DO - 10.1016/0003-9861(92)90094-D
M3 - Article
C2 - 1524443
AN - SCOPUS:0026668056
SN - 0003-9861
VL - 298
SP - 63
EP - 69
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -