Complete isomer-specific 1H and 13C NMR assignments of the heme resonances of rat liver outer mitochondrial membrane cytochrome b5.

Singly and doubly labeled delta-aminolevulinic acid derivatives were used to prepare rat liver outer mitochondrial membrane (OM) cytochrome b5 containing a 13C-labeled heme active site. A variety of NMR experiments, including HMBC and INADEQUATE in conjunction with the more commonly used HMQC, NOESY, and COSY, were conducted to make unambiguous assignments of protonated carbons and the quaternary pyrrole-alpha and -beta carbons in both isomeric forms of the paramagnetic active center of OM cytochrome b5. Because the long interpulse delays in the HMBC experiment have a detrimental effect on the detectability of fast relaxing paramagnetically affected resonances. INADEQUATE is proposed as the experiment of choice for assigning quaternary carbons in paramagnetic hemes with carefully chosen macrocycle labeling patterns. Furthermore, the applicability of the INADEQUATE experiment to paramagnetic heme active sites should be facilitated greatly by the availability of biosynthetic methods for producing isotopically labeled b-hemes and, more recently, isotopically labeled c-hemes.