Complete isomer-specific H-1 and C-13 NMR assignments of the heme resonances of rat liver outer mitochondrial membrane cytochrome b(5)

Singly and doubly labeled delta-aminolevulinic acid derivatives were used t o prepare rat liver outer mitochondrial membrane (OM) cytochrome b(5) conta ining a C-13-labeled heme active site. A variety of NMR experiments, includ ing HMBC and INADEQUATE in conjunction with the more commonly used HMQC, NO ESY, and COSY, were conducted to make unambiguous assignments of protonated carbons and the quaternary pyrrole-alpha and -beta carbons in both isomeri c forms of the paramagnetic active center of OM cytochrome bg Because the l ong interpulse delays in the HMBC experiment have a detrimental effect on t he detectability of fast relaxing paramagnetically affected resonances, INA DEQUATE is proposed as the experiment of choice for assigning quaternary ca rbons in paramagnetic hemes with carefully chosen macrocycle labeling patte rns. Furthermore, the applicability of the INADEQUATE experiment to paramag netic heme active sites should be facilitated greatly by the availability o f biosynthetic methods for producing isotopically labeled b-hemes and, more recently, isotopically labeled c-hemes.