2D 1H NMR investigations of paramagnetic proteins: The oxidized form of ferredoxin from clostridium pasteurianum.

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2D 1H NMR INVESTIGATIONS OF PARAMAGNETIC PROTEINS: THE OXIDIZED FORM OF FERREDOXIN FROM CLOSTRIDIUM PASTEURIANUM.

I. Bertini, L. Mesaori,R. Monmnn.i~A. Stmmf~~. Department of Chemistry, Universaty of Florence, 50121 Florence, Italy.

V. Gino Capponi,

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Clostridium Pasteurianum ferredoxin is a low molecular weight protein containing two cubaue Fe&4 clusters. The 1H NMR spectrum of the oxidized form of the protein, that is paramagnetic at room temperature, shows a substantial number of resonances isotropically shifted from the diamagnetic region. Their assi nment is important in order to monitor the magnetic properties o B the protein and it8 interactions with redox partners. In spite of the severe limitation8 caused by the short nuclear relaxation times, 2D NMR experiments are an appropriate tool to assign isotropically shifted si nals in metalloproteins. The experimental conditions have to be carefu piy adjusted in order to optimize cross-peak detection. In particular we show that 2D NOESY (Fig. 1A) and COSY magnitude (Fig. 1B experiment8 permit to reveal the dipolar and scalar connectivities o j the eight signals comprised between 20 and 10 ppm. The pattern of the observed crosspeaks allow8 us to assign all of them as Cys VII2 protons; their geminal partner8 are found in the region between 9.8 and 4.9 ppm. So, using the present approach we were able to assign all the ,0--CH2 protons of the eight metal bound cys teines. PPM t 4 I 8 10 12 14 10

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