On the function and mechanism of action of peroxidases

As a result of the adaptation of life to an aerobic environment, nature has evolved a panoply of metalloproteins for oxidative metabolism and protection against reactive oxygen species. Despite the diverse structures and functions of these proteins, they share common mechanistic grounds. An open-shell transition metal like iron or copper is employed to interact with O2 and its derived intermediates such as hydrogen peroxide to afford a variety of metal–oxygen intermediates. These reactive intermediates, including metal-superoxo, -(hydro)peroxo, and high-valent metal–oxo species, are the basis for the various biological functions of O2-utilizing metalloproteins. Collectively, these processes are called oxygen activation. Much of our understanding of the reactivity of these reactive intermediates has come from the study of heme-containing proteins and related metalloporphyrin compounds. These studies not only have deepened our understanding of various functions of heme proteins, such as O2 storage and trans...

Oxygen Activation and Radical Transformations in Heme Proteins and Metalloporphyrins

Metalloporphyrins undergo remarkable nonplanar distortions of the macrocycle that perturb the chemical and photochemical properties of these important protein cofactors. Further, the tertiary structure of the surrounding protein can manipulate these distortions as a means of regulating biological function. For cytochromes c, for example, an energetically unfavorable, conserved nonplanar distortion of the heme exists and likely plays a role in its electron- transfer function. The heme distortion is primarily of the ruffling (ruf, type (corresponding to the lowest frequency BI,-symmetry normal mode) in which the pyrroles are twisted about the metal-Npyrrole bond. This BI.-symmetry nonplanar distortion is commonly observed in metalloporphyrin crystal structures, as are the saddling (sad) B2,- symmetry distortion, waving (wau) E,-symmetry distortions, and doming (dom) Az,-symmetry distortion. Each of these nonplanar distortions is expected to result in unique alterations of the chemical and physical properties of the nominally planar porphyrin macrocycle. Symmetrical porphyrin substitution with tetrahedrally bonded atoms at the four meso bridging carbons generally results in the BI, ruffling distortion; therefore, we investigated a series of meso-tetrasubstituted porphyrins for which the substituents vary in size (methyl, ethyl, propyl, pentyl, isopropyl, cyclopropyl derivative lla, cyclohexyl, apopinenyl (lo), tert-butyl, adamantyl), increasing the steric crowding at the periphery. Molecular mechanics calculations show increasing degree of ruffling (CaNNCa angle for opposite pyrroles varies from 0 to 57") for this series of porphyrins, generally agreeing with the X-ray structures that are available. In addition, the frequencies of the structure-sensitive Raman lines decrease nonlinearly with increasing ruffling angle. The localization of the Bl, nonplanar distortion in only the C,-C, bond torsion (not the case for the B2" sad distortion) suggests a means by which the B1, distortion might be distinguished from other types of nonplanar distortion by using resonance Raman spectroscopy. Also, the size of the red shifts in the n - n* absorption bands depends on Ca-Cm torsion angle in a nonlinear fashion and the shift is accurately predicted by INDO/s molecular orbital calculations when the nonplanar structures obtained from molecular mechanics are used.

Ruffling in a Series of Nickel(II) meso-Tetrasubstituted Porphyrins as a Model for the Conserved Ruffling of the Heme of Cytochromes c.

Resonance Raman spectra of oxygenated and deoxygenated functional erythrocytes recorded using 785 nm laser excitation are presented. The high-quality spectra show a mixture of enhanced A1g, A2g, B1g, B2g, Eu and vinyl modes. The high sensitivity of the Raman system enabled spectra from four oxygenation and deoxygenation cycles to be recorded with only 18 mW of power at the sample over a 60-minute period. This low power prevented photo-/thermal degradation and negated protein denaturation leading to heme aggregation. The large database consisting of 210 spectra from the four cycles was analyzed with principal components analysis (PCA). The PC1 loadings plot provided exquisite detail on bands associated with the oxygenated and deoxygenated states. The enhancement of a band at 567 cm−1, observed in the spectra of oxygenated cells and the corresponding PC1 loadings plot, was assigned to the Fe–O2 stretching mode, while a band appearing at 419 cm−1 was assigned to the Fe–O–O bending mode based on previous studies. For deoxygenated cells, the enhancement of B1g modes at 785 nm excitation is consistent with vibronic coupling between band III and the Soret transition. In the case of oxygenated cells, the enhancement of iron-axial out-of-plane modes and non-totally symmetric modes is consistent with enhancement into the y,z-polarized transition ${\text{a}}_{{{\text{iu}}}} {\left( {\text{ $ \pi $ }} \right)} \to {\text{d}}_{{{\text{xz}}}} + {\text{O}}_{{\text{2}}} {\left( {{\text{ $ \pi $ }}_{{\text{g}}} } \right)}$ centered at 785 nm. The enhancement of non-totally symmetric B1g modes in oxygenated cells suggests vibronic coupling between band IV and the Soret band. This study provides new insights into the vibrational dynamics, electronic structure and resonant enhancement of heme moieties within functional erythrocytes at near-IR excitation wavelengths.

Tomoko Yamamoto

Papers

The Valence and Spin State of Iron in Oxyhemoglobin as Inferred from Resonance Raman Spectroscopy

Resonance Raman spectroscopy of cytochrome c oxidase and electron transport particles with excitation near the Soret band.

Resonance raman studies of a c type algal cytochrome. Deuterium shifts and a comparison with mammalian cytochrome c.