Quantitative measurement of small through-hydrogen-bond and through-space <Superscript>1 </Superscript>H- <Superscript>113 </Superscript>Cd and <Superscript>1 </Superscript>H- <Superscript>199 </Superscript>Hg J couplings in metal-substituted rubredoxin from <Emphasis Type="Italic">Pyrococcus furiosus </Emphasis>

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 -~t3Cd and I H-199Hg J couplings in metaJ-substituted rubredoxin (M, ~ 5.4 kDa) from Pyrococcusfuriosus. Couplings from cadmium to backbone amide protons that are hydrogen bonded to the Cys-S atoms directly bonded t o C d 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 199Hg are significantly larger and fall in the 0.4-4 Hz range.