NUCLEAR MAGNETIC RESONANCE STUDIES OF PLANT-TYPE FERREDOXINS (NMR)

TZE-MING CHAN, Purdue University

Abstract

Plant-type 2Fe(.)2S* ferredoxins have been studied by ('1)H and ('13)C NMR spectroscopy. Ferredoxins from six species (Anabaena variabilis, Anacystis nidulans, Spirulina maxima, Phytolacca americana, Spinacia oleracea, Leucaena glauca) were studied by ('1)H NMR. The majority of the ('13)C NMR studies were on enriched A. variabilis ferredoxin II (ul, 20% ('13)C), and a few natural abundance ('13)C NMR studies were on S. maxima ferredoxin II. Resonances in the aromatic region of the ('1)H spectra were assigned mainly by comparing the spectra and sequences of the six ferredoxins. The assignments were corroborated by Cr(NH(,3))(,6)('3+) binding experiments and chemical modifications of the proteins. Resonances in the aromatic region of the ('13)C spectra were assigned by cross-correlation with the assigned ('1)H resonances: the protonated carbons were assigned by heteronuclear (('13)C,('1)H) two-dimensional chemical shift correlated spectroscopy, and the non-protonated carbons were assigned by coherent selective decoupling experiments. The single Cr(NH(,3))(,6)('3+) binding site on ferredoxin discovered by Armstrong and coworkers {J. Chem. Soc. Chem. Commum. (1978) 1102-1103} was found to be near Tyr('83) and Tyr('25), since resonances in the ('1)H and ('13)C spectra due to these residues were selectively broadened. There are several negatively-charged groups around this site, which can interact electrostatically with the positively-charged chromium(III) complex. The spin-lattice relaxation times for several non-protonated carbons near to the 2Fe(.)2S* center were found to be much shorter in oxidized ferredoxin than in reduced ferredoxin. Also there is more intensity in the aromatic region of the spectra of reduced proteins than in those of oxidized proteins. These results imply that the electron spin relaxation time (T(,e)) for the iron atoms in the reduced ferredoxin is shorter than that for those in the oxidized ferredoxin. The ferredoxin-ferredoxin NADP('+) oxidoreductase complex was studied by ('13)C NMR. At least three glutamic acid residues on the ferredoxin molecules were found to be at or near the contact region between the proteins. The ferredoxin molecule does not undergo a major conformational change when bound to the oxidoreductase.

Degree

Ph.D.

Subject Area

Biochemistry

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