QUANTITATIVE MEASUREMENT OF SMALL THROUGH-HYDROGEN-BOND AND THROUGH-SPACE H-1-CD-113 AND H-1-HG-199 J-COUPLINGS IN METAL-SUBSTITUTED RUBREDOXIN FROM PYROCOCCUS-FURIOSUS

A method is described for measurement of small unresolvable heteronuclear J couplings. The method is based on quantitative analysis of a phase-purged heteronuclear spin-echo difference spectrum, and is demonstrated for measuring H-1-Cd-113 and H-1-Hg-199 J couplings in metal-substituted rubredoxin (M(r) approximately 5.4 kDa) from Pyrococcus furiosus. Couplings from cadmium to backbone amide protons that are hydrogen bonded to the Cys-S atoms directly bonded to Cd vary from smaller than 0.3 to 1.8 Hz; a 'through-space' coupling between Cd and the protons of an alanine methyl group was measured to be 0.3 Hz. Couplings to Hg-199 are significantly larger and fall in the 0.4-4 Hz range.