Hydrogen-1 nuclear magnetic resonance investigation of high-potential iron-sulfur proteins from Ectothiorhodospira halophila and Ectothiorhodospira vacuolata: a comparative study of hyperfine-shifted resonances.

Proton NMR spectra of the oxidized and reduced forms of high-potential iron-sulfur proteins (HiPIPs) were recorded at 200 MHz. The proteins studied were the HiPIPs I and II from Ectothiorhodospira halophila and Ectothiorhodospira vacuolata. Hyperfine-shifted peaks in spectra of the oxidized proteins were assigned to some of the protons of the cysteinyl ligands and aromatic residues at the active site on the basis of their chemical shifts, longitudinal relaxation times, and temperature-dependent behavior. The cysteinyl C beta-H protons were found to resonate downfield (about 100 ppm) and the C alpha-H protons upfield (about-25 ppm). This hyperfine shift pattern is consistent with the observed isotropic shift being contact in origin; it probably results from a pi-spin-transfer mechanism. The large magnitudes of the chemical shifts of peaks assigned to aromatic residues suggest that these residues interact with the iron-sulfur cluster via pi-pi overlap. Some of the hyperfine-shifted peaks observed in water were found to disappear in 2H2O solution. Such resonances probably arise from exchange-labile hydrogens of amino acid residues directly hydrogen bonded to the iron-sulfur cluster. In the case of HiPIPs I and II from E. vacuolata, whose spectra are similar except for the number of such peaks, the relative number of hydrogen bonds inferred to be present in the oxidized and reduced proteins qualitatively explains the difference between their midpoint redox potentials. On the other hand, for E. halophila HiPIPs I and II, consideration of the inferred number of hydrogen bonds alone fails to predict the sign of the difference between their midpoint redox potentials.(ABSTRACT TRUNCATED AT 250 WORDS)