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

Abstract

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 Å 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.

Isolation and characterization of a microperoxidase-8 with a modified histidine axial ligand

“…4c) shows two NOEs from the saturated peak to, respectively, the b-meso and the 4-aH protons. A NOE interaction between the b-meso and the 4-aH protons has been reported on microperoxidase-11 (having three extra C-terminal residues when compared to Fe(III)MP-8) [66]. Based on these NOEs, the resonance at 16.2 ppm is found consistent with the assignment to the non-exchangeable proton Hd2 of the axial histidine ligand.…”

Peroxidase model electrodes: Self-mediation of heme peptide multilayer-modified electrodes and its application to biosensing with adjustable dynamic range

“…Since the e value of HP monomer-unit becomes smaller when dimers and oligomers are formed, an actual C value would be larger. The volume of the hydrated HP molecule has been reported to be 796 · 10 À23 cm 3 [34]. Assuming that the HP molecule is spherical and hexagonally packed on the electrode surface, the C value of the HP monolayer was calculated to be 3.5 · 10 À11 mol cm À2 .…”

“…Since HP molecules were immobilized in the film, D 0 was 0. As HP molecule was assumed to be 796 · 10 À23 cm 3 (V HP ) of a sphere [34], o was calculated to be 2.48 nm (V HP = 4p(o/2) 3 /3). The k ex values of the anodic reaction at +800 mV were calculated to be 1-3 · 10 2 and 1-3 · 10 3 M À1 s À1 for two and one electron reactions, respectively.…”

Small Molecule Directed Aggregation of a Heme Peptide on Gold: An STM Study

“…This result is reproducible and contrasts with the solution behavior of MP11 with exogenous ligands, such as CN -, imidazole, and ammonia where the heme peptide is predominantly monomeric. 10,11 Based upon the apparent dimensions (∼6 nm width, ∼15 nm length, ∼ 2.2 nm height) of the nano-e ´pis (Figure 1b), it is likely that their discernible subunits are either dimers or trimers, although at this time the mode of aggregation is unclear.…”