Redox infrared markers of the heme and axial ligands in microperoxidase: bases for the analysis of c-type cytochromes

Abstract

Structural changes accompanying the change in the redox state of microperoxidase-8 (MP8), the heme-octapeptide obtained from cytochrome c, and its complexes with (methyl)imidazole ligands were studied by electrochemically induced Fourier transform IR (FTIR) difference spectroscopy. To correlate with confidence IR modes with a specific electronic state of the iron, we used UV-vis and electron paramagnetic resonance spectroscopy to define precisely the heme spin state in the samples at the millimolar concentration of MP8 required for FTIR difference spectroscopy. We identified four intense redox-sensitive IR heme markers, nu38 at 1,569 cm(-1) (ox)/1,554 cm(-1) (red), nu42 at 1,264 cm(-1) (ox)/1,242 cm(-1) (red), nu43 at 1,146 cm(-1) (ox), and nu44 at 1,124-1,128 cm(-1) (ox). The intensity of nu42 and nu43 was clearly enhanced for low-spin imidazole-MP8 complexes, while that of nu44 increased for high-spin MP8. These modes can thus be used as IR markers of the iron spin state in MP8 and related c-type cytochromes. Moreover, one redox-sensitive band at 1,044 cm(-1) (red) is attributed to an IR marker specific of c-type hemes, possibly the delta(CbH3)(2,4) heme mode. Other redox-sensitive IR bands were assigned to the MP8 peptide backbone and to the fifth and sixth axial heme ligands. The distinct IR frequencies for imidazole (1,075 cm(-1)) and histidine (1,105 cm(-1)) side chains in the imidazole-MP8 complex allowed us to provide the first direct determination of their pKa at pH 9 and 12, respectively.

Recombinant expression and redox properties of trihemecmembrane-bound quinol peroxidase

“…Thus, relatively high E m value of the lowest potential of the heme would be explained by the fact that the amino acid sequence of QPO that apparently lacks distal ligand of the heme in the middle portion. This idea would be supported by the previous observation showing five‐coordinated heme c exhibits higher E m value than that of six‐coordinated heme c (Marboutin et al , 2006). However, although five‐coordinated heme c have lower extinction coefficient (Marboutin et al , 2006), the relative spectral contribution of the each heme in QPO was nearly same.…”

“…This idea would be supported by the previous observation showing five‐coordinated heme c exhibits higher E m value than that of six‐coordinated heme c (Marboutin et al , 2006). However, although five‐coordinated heme c have lower extinction coefficient (Marboutin et al , 2006), the relative spectral contribution of the each heme in QPO was nearly same. Further experiment is needed to address the coordination of heme in QPO.…”

C‐Type Cytochromes Wire Electricity‐Producing Bacteria to Electrodes

“…In addition to the amide I related bands, a number of differential bands were detected upon full oxidation of the cell molecules ( Figure S1b in the Supporting Information). The assignment of some of them remains unknown, but major ones at 1602, 1573, 1385, and 1322 cm À1 and minor ones at 1554, 1479, 1406, 1398, 1243, and 1148 cm À1 have been reported in relation to Cyt-C [13,19] or Microperoxidase 8, the octapeptide containing the heme group in Cyt-C. [20] Most of these cytochrome-related bands were also observed to reverse during the redox transition ( Figure S3 in the Support-ing Information). Some assigned to different vibrational modes of the heme ring (1602,1573,1385,1479,1406,1243, and 1148 cm À1 ), [20] probably reflect the high number of heme moieties borne by cytochromes interacting with the gold surface.…”

“…[19] Importantly, the amide III band splits into two minor bands at 1400 and 1381 cm À1 , that jointly with the one at 1145 cm À1 , could also be related to reported vibrations of the heme ring. [20] Heterogeneous electron transport requires active molecules close enough to the electrode to facilitate the tunneling of electrons, thus ensuring the occurrence of the reaction within the distance range of the SEIRA evanescent field. Taking advantage of this fact, we are able to detect conformational changes in the "electrode-reducing" molecules.…”

Metal-histidine-glutamate as a regulator of enzymatic cycles: a case study of carbonic anhydrase

“…8,9 Several experimental studies based on NMR, 10 resonance Raman 11 or Fourier transform infrared [12][13][14] spectroscopy suggest the formation of iron-imidazolate moiety in heme and non heme proteins. Notably, the presence of the Fe-His-Asp/Glu triad, associated with the low pK a value of the metal-bound histidine, 15 is consistent with a deprotonated His ligand. In heme proteins, this is associated with the charge relay mechanism where the hydrogen bond network around the iron-bound histidine tunes the charge donated by the His to the Fe, and stabilize oxidation states greater than Fe(III).…”