1H, 15N and 13C NMR resonance assignment, secondary structure and global fold of the FMN-binding domain of human cytochrome P450

The FMN-binding domain of human NADPH-cytochrome P450 reductase,corresponding to exons 3-;7, has been expressed at high level in anactive form and labelled with ^sup 13^C and ^sup 15^N. Mostof the backbone and aliphatic side-chain ^sup 1^H, ^sup 15^Nand ^sup 13^C resonances have been assigned using heteronucleardouble- and triple-resonance methods, together with a semiautomaticassignment strategy. The secondary structure as estimated from the chemicalshift index and NOE connectivities consists of six α-helices and fiveβ-strands. The global fold was deduced from the long-range NOEsunambiguously assigned in a 4D ^sup 13^C-resolved HMQC-NOESY-HMQCspectrum. The fold is of the alternating α/β type, with the fiveβ-strands arranged into a parallel β-sheet. The secondarystructure and global fold are very similar to those of the bacterialflavodoxins, but the FMN-binding domain has an extra short helix in place ofa loop, and an extra helix at the N-terminus (leading to the membrane anchordomain in the intact P450 reductase). The experimental constraints werecombined with homology modelling to obtain a structure of the FMN-bindingdomain satisfying the observed NOE constraints. Chemical shift comparisonsshowed that the effects of FMN binding and of FMN reduction are largelylocalised at the binding site.[PUBLICATION ABSTRACT]