Structural Characterization of Cytochrome c Peroxidase by Resonance Raman Scattering

Peroxidase to Cytochrome b Type Transition in the Active Site of Heme-Bound Amyloid β Peptides Relevant to Alzheimer’s Disease

“…The low-frequency region in the rR spectrum (180–500 cm –1 ) for Fe II -heme proteins exhibits iron–ligand stretching vibrations along with Fe–N­(pyrrole) in-plane and out-of-plane frequency modes. The rR spectrum of the ferrous 1:1 heme-Aβ complex shows a vibration at 241 cm –1 (Figures A and S4A), consistent with the Fe–His stretching mode observed in peroxidases. , This provides direct evidence for His coordination in heme–Aβ complexes. , …”

“…The ν 2 region exhibits a broad band at 1575 cm –1 (Figure B). These indicate the presence of a predominantly five-coordinate high-spin ferric heme site along with six-coordinate low-spin and six-coordinate high-spin heme centers. − With an increase in the pH, the rR spectrum shows an increase in the intensity of the 1492 and 1484 cm –1 bands and a decrease in the intensity of the 1505 cm –1 band reflecting an increase in the population of high-spin components and a decrease in the population of low-spin component (Figures B,D and S1A,B). The charge-transfer band at 632 nm in the absorption spectrum at neutral pH blue shifts to 605 nm at pH 9.5 (Figures A and S1A), suggesting conversion of a H 2 O-bound heme species to its corresponding OH – -bound form. , This blue shift of the charge-transfer band validates the presence of the six-coordinate high-spin heme center, which was previously proposed to be a His-bound heme with a trans axial water-derived ligand at the distal pocket. , However, the presence of the water-derived ligand at the sixth position either at high or neutral pH could not be confirmed by an isotope labeling study (using D 2 O 16 or H 2 O 18 as the solvent instead of H 2 O 16 ; Figures B and S2).…”

“…The appearance of the ν 38 frequency at 1548 cm –1 in the rR spectra also indicates the growth of a low-spin species. Note that the α- and β-band positions in the absorption spectrum are characteristic of a bis-His (two axial His ligands of heme–Aβ) six-coordinate low-spin active site akin to those found in cytochrome b type proteins, neuroglobin and cytoglobin. ,,− Upon dilution of heme–Aβ complex solutions with buffer solutions maintaining the same pH, the high-spin active sites (five- and six-coordinate) are gradually formed. Thus, these data indicate the presence of an equilibrium between the high- and low-spin heme–Aβ species in the same active site under physiological conditions ,− (Scheme ).…”

Regenerative silver nanoparticles for SERRS investigation of metmyoglobin with conserved heme pocket

“…Previous studies of metMb on a silver surface signify that direct contact between them resulted in the loss of a distal water molecule from the heme pocket leading to the denaturation and subsequent appearance of a signal corresponding to a 5 coordinated high spin heme structure. 31,44,49,50 Additionally, increasing the incubation time between enlarged Ag@citrate NPs and metMb resulted in an increased intensity of the 1494 cm 21 band indicating the further removal of an axial ligand i.e., the water molecule. Thus, in analogy to previous reports the native structure of metMb is disturbed upon incubation with enlarged Ag@citrate NPs.…”

“…Marker bands for metMb and their corresponding assignments are listed in Table 1. 1,31,[40][41][42][43][44] The n 4 band is sensitive to the oxidation state of the heme iron centre and generally appears between 1368-1377 cm 21 for ferric heme and 1344-1364 cm 21 for ferrous heme. In the present study, the n 4 band appearing at 1374 cm 21 corresponds to the ferric heme centre as expected for metMb.…”