19F NMR Studies of Fluorine-Labeled Chromatium vinosum High-Potential Iron Protein

Abstract

Fluorine-labeled analogues of Chromatium vinosum high-potential iron protein have been investigated by 19F NMR spectroscopy. Fluorine-19 resonances have been assigned, and chemical shift variations, relaxation times, and temperature dependencies have been determined. Observed changes in fluorine chemical shifts and relaxation times following oxidation of the [Fe4S4] cofactor appear to reflect structural perturbation of the protein backbone and side chains, rather than variations in the paramagnetism of the cluster. Fluorine-19 NMR provides a probe of redox-dependent conformational change in electron-transfer proteins, which may also be of value for structural characterization of mutants. Evaluation of H2O/D2O solvent isotope effects on 19F chemical shifts reflects solvent accessibility to various protein domains, while measurement of 19F relaxation times affords a convenient test of the relative contribution of cross-relaxation to magnetization decay. For HiPIP, the results reported herein indicate that the cross-relaxation contribution to the longitudinal relaxation (T1) is relatively small for both the oxidized and reduced states. Unusual temperature dependencies and fast relaxation times for the 19F resonances of 3-F-Phe66 and 3-F-Tyr19 labeled HiPIP support a close interaction of these two residues with the iron−sulfur cluster.