CHARACTERIZATION OF HYPERFINE-SHIFTED H-1 RESONANCES IN OXIDIZED AND REDUCED PUTIDAREDOXIN, AN FE2S2 FERREDOXIN FROM PSEUDOMONAS-PUTIDA

Hyperfine-shifted H-1 resonances in the two accessible oxidation states of putidaredoxin (Pdx), an Fe2S2 ferredoxin from Pseudomonas putida, were observed and their temperature dependence and relaxation times measured using rapid-recycle NMR methods. As with other Fe2S2 ferredoxins, the hyperfine-shifted resonances observed in oxidized (Fe3+-Fe3+) Pdx exhibit anti-Curie behavior, their signals moving away from the diamagnetic region with increasing temperature. The hyperfine-shifted resonances of reduced Pdx (Fe3+-Fe2+) exhibit Curie-type behavior, and closely resemble the appearance and behavior of the hyperfine shifted resonances of human ferredoxin (HFdx), with which Pdx shares significant sequence and functional homology. Electron self-exchange is slow on the H-1 chemical shift time-scale, as half-reduced samples of Pdx exhibit hyperfine-shifted resonances characteristic of both forms.