In paramagnetic heme proteins, it is often problematic to make the reduced (usually diamagnetic) state lack isotropic shifts proton resonance assignments for heme substituents that do not and are therefore difficult to examine by NMR spectroscopy. have large isotropic shifts and consequently lie under the large Observation of these resonances is important in order to envelope of polypeptide resonances. Furthermore, assignments study structure-function relationships in electron-transfer that would normally be performed with the aid of HMBC experiheme proteins, which function by shuttling their heme iron ments in diamagnetic molecules can prove difficult in the active between ferric and ferrous oxidation states. (c) Efficient site of paramagnetic heme proteins if T 01 2 ú 2 J CH . To circumvent spin-spin relaxation often makes through-bond proton-prothis problem, a new method is presented to selectively detect 1 H ton correlations in COSY or TOCSY experiments unobservin 1 H n -13 C-13 C fragments biosynthetically introduced into the able (T 01 2 ú J HH ) (2, 6). In fact, it has been proposed that active site of heme proteins. The pulse sequence combines wellthe COSY cross peaks observed in paramagnetic systems known building blocks such as INEPT to transfer 1 H spin magnetization to bonded 13 C nuclei, followed by INADEQUATE to gener-arise from dipolar coupling and Curie spin-nuclear spin ate 13 C-13 C double-quantum coherence that is selected with relaxation (7, 8). pulsed field gradients, and finally reverse-INEPT to transfer mag-
One strategy designed to circumvent some of these probnetization back to 1 H nuclei for subsequent observation. The new lems involves the selective 13 C enrichment of heme substitu-1 H n -13 C-13 C edited experiment takes advantage of the relatively ents and their subsequent observation by 13 C NMR spectroslarge values of 1 J CH and 1 J CC , avoiding the long interpulse delays copy. We have recently reported a biosynthetic method for in HMBC that compromise the detectability of rapidly relaxing the expression of 13 C-labeled heme cytochrome b 5 (9), thus nuclei. The potential applicability of the pulse sequence is demonfacilitating NMR experiments such as HMQC and HSQC strated by its contribution to the unambiguous assignment of the (10-14) that rely on the heteronuclear through-bond concarbonyl carbons in the heme propionates of ferricytochrome b 5 .
nectivity and larger scalar coupling constant 1 J CH . However,