3D structure of microperoxidase-11 by NMR and molecular dynamic studies

Microperoxidase-11 (MP11) is an undecapeptide, obtained from enzymatic cleavage of cytochrome c, which still exhibits peroxidase activity. It retains residues 11–21 of the protein, the heme-c group and His18 as fifth ligand at the iron atom. We studied MP11 in the paramagnetic, Fe(III) low-spin state by NMR and constrained molecular dynamics (MD). Well-resolved 1H and 13C spectra were obtained in water–trifluoroethanol and water–methanol mixtures with different axial ligands to the iron, i.e. imidazole, NH3, OH, CN, at different temperatures from −15 to +25 °C. All the protons of the heme and the peptide moiety were assigned by using TOCSY and NOESY experiments. Some stereospecific assignments were also performed and eight coupling constants between the amide and the α-protons were measured. Seventy-one inter-residue and heme peptide and 40 intra-residue NOE interactions were translated into inter-proton distances. Ten structures satisfying these distance constraints to within ±0.4 A were obtained and showed that almost the whole peptide (segment 12–19) can form a right-handed helix. The envelope structure of the peptide around the heme is well defined based on a number of NOE interactions between heme and peptide protons. Variable-temperature experiments were performed in order to obtain information on the protons affected by the paramagnetic shift. An inverted temperature dependence (Curie law) was observed for the heme and meso protons, except for 3-Me, when the sixth axial ligand is NH3 or imidazole. The pattern of 13C hyperfine shifts of heme methyl groups, similar in MP11 and cytochrome c, suggests that the orientation of His ring along the α–γ meso axis is conserved in MP11, which is consistent with MD calculations. Copyright © 2000 John Wiley & Sons, Ltd.