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.

We report the setup of an electrochemical cell with chemical-vapor deposition diamond windows and the use of a Bruker 66 SX FTIR spectrometer equipped with DTGS and Si-bolometer detectors and KBr and mylar beam splitters, to record on the same sample, FTIR difference spectra corresponding to the structural changes associated with the change in redox state of active sites in proteins in the whole 1800-50 cm -1 region. With cytochrome c we show that reliable reducedminus-oxidized FTIR difference spectra are obtained, which correspond to single molecular vibrations. Redox-sensitive IR modes of the cytochrome c are detected until 140 cm -1 with a good signal to noise. This new setup is promising to analyze the infrared spectral region where metal-ligand vibrations are expected to contribute and to extend the analysis of vibrational properties to metal sites or redox states not accessible to (resonance) Raman spectroscopy. # 2006 Wiley Periodicals, Inc.

FTIR difference spectroscopy, notably in the far-IR domain, is appealing for the analysis of hemoproteins, since it permits us to directly probe the properties of the heme and its ligands but also those of aminoacids remote from the heme. We recently set a thin path-length electrochemical cell with diamond windows allowing the far-IR analysis of proteins in aqueous solutions using FTIR difference spectroscopy (Berthomieu, C,; Marboutin, L.; Dupeyrat, F.; Bouyer, P. Biopolymers 2006 82, 363-367). In this study, we used this cell to identify redox-sensitive low-frequency IR modes of imidazole complexes of Fe-protoporphyrin IX and microperoxidase-8 and analyzed the pH dependence of these modes. The far-IR bands of the heme and the axial imidazole ligands were assigned using (15)N(2)-, and d(3)-imidazole isotopic substitution, as well as imidazole substitution by 4(5)-methylimidazole. Internal modes of the axial histidine and imidazole ligands were identified in the 670-580 cm(-1) region, which are sensitive to the iron coordination (five-coordinated high-spin heme or six-coordinated low-spin heme) and the protonation states of the axial ligands. We showed that deformation modes of the heme pyrroles dominate the 420-370 cm(-1) region of the difference spectra. These modes were highly sensitive to the coordination and redox states of the heme iron and the conformation of the tetrapyrrole. While no nu(as)(Fe-axial ligand) IR mode was detected in the difference spectra of the neutral imidazole complexes of Fe-protoporphyrin and microperoxidase, a new mode at 312 and 334 cm(-1) was found specific of the imidazolate complexes of Fe(3+)-protoporphyrin and Fe(3+)-microperoxidase-8, respectively. On the basis of isotope shifts observed upon ligand deuteration, this band was assigned to a mode mixing the asymmetric stretching of the axial bonds with an internal deformation of the imidazolate rings. These data set the bases for the analysis of the IR low-frequency modes of hemoproteins, and specifically the electronic properties of the heme axial histidine ligands.

Vibrations of the metal active site of the Cu,Zn-superoxide dismutase enzyme were analyzed by far-infrared difference spectroscopy and theoretical normal mode calculation. Both electrochemically triggered Cu(I) and Cu(II) redox states show well-defined infrared vibrational modes, notably modes of the histidine ligands, the Cu(II)-His(61)-Zn(II) bridge and of the water pseudo-ligand.

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