F-19 NMR-STUDIES OF FLUORINE-LABELED CHROMATIUM-VINOSUM HIGH-POTENTIAL IRON PROTEIN

Fluorine-labeled analogues of Chromatium vinosum high-potential iron p rotein have been investigated by F-19 NMR spectroscopy. Fluorine-19 re sonances have been assigned, and chemical shift variations, relaxation times, and temperature dependencies have been determined, Observed ch anges in fluorine chemical shifts and relaxation times following oxida tion of the [Fe4S4] cofactor appear to reflect structural perturbation of the protein backbone and side chains, rather than variations in th e paramagnetism of the cluster. Fluorine-19 NMR provides a probe of re dox-dependent conformational change in electron-transfer proteins, whi ch may also be of value for structural characterization of mutants. Ev aluation of H2O/D2O solvent isotope effects on F-19 chemical shifts re flects solvent accessibility to various protein domains, while measure ment of F-19 relaxation times affords a convenient test of the relativ e contribution of cross-relaxation to magnetization decay. For HiPIP, the results reported herein indicate that the cross-relaxation contrib ution to the longitudinal relaxation (T-1) is relatively small for bot h the oxidized and reduced states, Unusual temperature dependencies an d fast relaxation times for the F-19 resonances of 3-F-Phe66 and 3-F-T yr19 labeled HiPIP support a close interaction of these two residues w ith the iron-sulfur cluster.