Modulation of the flavin-protein interactions in NADH peroxidase and mercuric ion reductase: a resonance Raman study.

NADH peroxidase (Npx) and mercuric ion reductase (MerA) are flavoproteins belonging to the pyridine nucleotide:disulfide oxidoreductases (PNDO) and catalyzing the reduction of toxic substrates, i.e., hydrogen peroxide and mercuric ion, respectively. To determine the role of the flavin adenine dinucleotide (FAD) in the detoxification mechanism, the resonance Raman (RR) spectra of these enzymes under various redox and ligation states have been investigated using blue and/or near-UV excitation(s). These data were compared to those previously obtained for glutathione reductase (GR), another enzyme of the PNDO family, but catalyzing the reduction of oxidized glutathione. Spectral differences have been detected for the marker bands of the isoalloxazine ring of Npx, MerA, and GR. They provide evidence for different catalytic mechanisms in these flavoproteins. The RR modes of the oxidized and two-electron reduced (EH2) forms of Npx are related to very tight flavin-protein interactions maintaining a nearly planar conformation of the isoalloxazine tricycle, a low level of H-bonding at the N1/N5 and O2/O4 sites, and a strong H-bond at N3H. They also indicate minimal changes in FAD structure and environment upon either NAD(H) binding or reduction of the sulfinic redox center. All these spectroscopic data support an enzyme functioning centered on the Cys-SO-/Cys-S- redox moiety and a neighbouring His residue. On the contrary, the RR data on various functional forms of MerA are indicative of a modulation of both ring II distortion and H-bonding states of the N5 site and ring III. The Cd(II) binding to the EH2-NADP(H) complexes, biomimetic intermediates in the reaction of Hg(II) reduction, provokes important spectral changes. They are interpreted in terms of flattening of the isoalloxazine ring and large decreases in H-bonding at the N5 site and ring III. The large flexibility of the FAD structure and environment in MerA is in agreement with proposed mechanisms involving C4a(flavin) adducts.

Redox effects on the coordination geometry and heme conformation of bis(N-methylimidazole) complexes of superstructured Fe-porphyrins. A spectroscopic study.

Electronic absorption, electron paramagnetic resonance (EPR), and Soret-excited resonance Raman (RR) spectra are reported for bis(N-alkylimidazole) complexes of various iron(III)-"basket-handle" (Fe(III)BHP(+)) and "picket-fence" (Fe(III)PFP(+)) porphyrins in methylene chloride. The Fe(III)BHP(+) derivatives consist of four cross-trans (CT) and two adjacent-cis (AC) -linked in which the composition and the length of the handles are variable (CT Fe(III)[(C(11)Im)(2)(+)], CT and AC Fe(III)[((C(4))(2)phi)(2)](+), CT Fe(III)[((C(3))(2)phi)(C(12))](+), CT and AC Fe(III)[((C(3))(2)phi)(2)](+)). The meso-alphaalpha betabeta and meso-alphabeta alphabeta atropisomers of Fe(III)-tetrakis(o-pivalamidophenyl)-porphyrins represents the Fe(III)PFP(+) derivatives (Fe(III)alphaalpha betabeta-T(piv)PP(+) and Fe(III)alphabeta alphabeta-T(piv)PP(+), respectively). The absorption and RR data obtained for these ferric compounds were compared to those previously published for the homologous ferrous complexes (Picaud, T., Le Moigne, C., Loock, B., Momenteau, M. and Desbois, A. J. Am. Chem. Soc. 2003, 125, 11616 and Le Moigne, C., Picaud, T., Boussac, A., Loock, B., Momenteau, M. and Desbois, A. Inorg. Chem. 2003, 42, 6081). The Soret band position of the eight investigated ferric compounds is observed between 417 and 424 nm, indicating that none of the complexes possesses a planar heme. The EPR spectra show that most of the Fe(III)BHP(+) complexes and all the Fe(III)PFP(+) complexes are rhombic B-type hemichromes (g(max) = 2.86-2.96). Notable exceptions concern the bis(N-methylimidazole) complexes of two CT Fe(III)BHP(+). The Fe(III)BHP(+) with the shortest handles (Fe(III)[((C(3))(2)phi)(2)](+)) exhibits a g value at 2.80. When the handles are lengthened by two methylene units (Fe(III)[((C(3))(2)phi)(2)](+)), the EPR spectrum corresponds to a mixture of two "highly anisotropic low-spin " or "large g(max)" type I EPR signals, a major species at g = 3.17 and a minor species at g = 3.77. All these EPR data were converted in terms of dihedral angle formed by the rings of the axial ligands. The RR spectra of the Fe(III)BHP(+) and Fe(III)PFP(+) complexes exhibited variable frequencies for the structure-sensitive nu(2) and nu(8) lines (1558-1563 cm(-1) and 386-401 cm(-1), respectively). In considering the ability of the different superstructures to stabilize particular out-of-plane distortions, this vibrational information was analyzed in terms of heme structure through changes in core size and Fe-N(pyrrole) bond length, in relation to changes in coordination geometry. The bis(N-methylimidazole) complex of Fe(III)[((C(3))(2)phi)(2)](+) was found to be the most distorted with a strongly ruffled tetrapyrrole. Because of a handle asymmetry, the heme conformation of the bis(N-methylimidazole) complex of Fe(III)[((C(3))(2)phi)(C(12))](+) was deduced to be a composition of ruffled and domed structures. The heme structure of the other complexes is a mixture of ruffled and saddled or ruffled and waved conformations. Taking into account our previous data on the ferrous series, this investigation provides information about the reorganization of the heme structure upon iron oxidation. The general trend is a decrease of either the core-size, or the Fe-N(pyrrole) bond length, or both. However, we demonstrated that the heme superstructures precisely control the nature and the extent of the tetrapyrrole reshaping. These results point out similar possible effect in the heme proteins, considering both an analogy between porphyrin superstructures and amino acids forming the heme sites and the diversity of the heme environments in the proteins.

Interaction of glutathione reductase with heavy metal: the binding of Hg(II) or Cd(II) to the reduced enzyme affects both the redox dithiol pair and the flavin.

To determine the inhibition mechanism of yeast glutathione reductase (GR) by heavy metal, we have compared the electronic absorption and resonance Raman (RR) spectra of the enzyme in its oxidized (Eox) and two-electron reduced (EH2) forms, in the absence and the presence of Hg(II) or Cd(II). The spectral data clearly show a redox dependence of the metal binding. The metal ions do not affect the absorption and RR spectra of Eox. On the contrary, the EH2 spectra, generated by addition of NADPH, are strongly modified by the presence of heavy metal. The absorption changes of EH2 are metal-dependent. On the one hand, the main flavin band observed at 450 nm for EH2 is red-shifted at 455 nm for the EH2-Hg(II) complex and at 451 nm for the EH2-Cd(II) complex. On the other hand, the characteristic charge-transfer (CT) band at 540 nm is quenched upon metal binding to EH2. In NADPH excess, a new CT band is observed at 610 nm for the EH2-Hg(II)-NADPH complex and at 590 nm for EH2-Cd(II)-NADPH. The RR spectra of the EH2-metal complexes are not sensitive to the NADPH concentration. With reference to the RR spectra of EH2 in which the frequencies of bands II and III were observed at 1582 and 1547 cm-1, respectively, those of the EH2-metal complexes are detected at 1577 and 1542 cm-1, indicating an increased flavin bending upon metal coordination to EH2. From the frequency shifts of band III, a concomitant weakening of the H-bonding state of the N5 atom is also deduced. Taking into account the different chemical properties of Hg(II) and Cd(II), the coordination number of the bound metal ion was deduced to be different in GR. A mechanism of the GR inhibition is proposed. It proceeds primarily by a specific binding of the metal to the redox thiol/thiolate pair and the catalytic histidine of EH2. The bound metal ion then acts on the bending of the isoalloxazine ring of FAD as well as on the hydrophobicity of its microenvironment.

Absorption and resonance Raman investigations of ligand rotation and nonplanar heme distortion in bis-base low-spin iron(II)-tetrakis(o-pivalamidophenyl)porphyrin complexes.

The absorption and resonance Raman (RR) spectra of the bis-N-methylimidazole, bis-1,5-dicyclohexylimidazole, and bis-pyridine complexes of the meso-alphaalphabetabeta and meso-alphabetaalphabeta atropisomers of Fe(II)-tetrakis(o-pivalamidophenyl)porphyrins (Fe(II)TpivPP) were obtained in methylene chloride. The different spatial arrangements of the o-pivalamide pickets in these two Fe(II)TpivPP compounds are expected to control the absolute and relative positions of the axial ligand rings with respect to the Fe-N(pyrrole) bonds. In particular, the spectroscopic data obtained for the bis-N-methylimidazole and bis-dicyclohexylimidazole complexes of the Fe(II)[alphabetaalphabeta-TpivPP] derivative showed the most important differences. Redshifts of the B and Q absorption bands (+ 4-5 nm) as well as an upshift of the low frequency nu(8) RR mode (+ 5 cm(-)(1)) were observed. No shift of the skeletal high frequency modes was detected. These spectral effects were associated with a change in relative position of the axial imidazole rings from nearly parallel in the bis-N-methylimidazole complex to nearly perpendicular in the bis-dicyclohexylimidazole complex. On the basis of stereochemical considerations as well as previous spectroscopic investigations, the data were interpreted in terms of change in porphyrin structure from planar to saddled. Complementing to a parallel study on bis-base Fe(II) "basket handle" porphyrin complexes, this spectroscopic investigation provides an additional means to distinguish planar, ruffled, and saddled conformations for ferrous hemes included in proteins.

Nonplanar distortions of bis-base low-spin iron(II)-porphyrinates: absorption and resonance Raman investigations of cross-trans-linked iron(II)-basket-handle porphyrin complexes.

Electronic absorption and Soret-excited resonance Raman (RR) spectra are reported for bis-N-alkylimidazole and bis-pyridine complexes of various cross-trans-linked iron(II)-"basket-handle" porphyrins (Fe(II)-BHP) in methylene chloride. These compounds enable us to characterize the spectroscopic properties of ruffled six-coordinated low-spin Fe(II)-porphyrin complexes. The visible absorption spectra show that the Q and B bands are progressively red-shifted when the handles are shortened and/or when the steric hindrance of the axial ligands is increased. This effect is accompanied by both a decrease in RR frequency of the nu(2) mode and an increase in frequency of the nu(8) and nu(s)(Fe-ligand(2)) modes. More precisely, an inverse linear correlation is found between the frequencies of the nu(2) and nu(8) modes. For each ligation state, the positions of the absorption bands are also linearly correlated with the frequency of the nu(2) or nu(8) mode. All of these spectroscopic data reveal that the degree of ruffling of the Fe(II)-BHP complexes is increased by the N-methylimidazole --> pyridine axial substitutions, presumably because the mutual steric strains between the axial ligand rings, the porphyrin macrocycle and the porphyrin handles are increased. The present study provides a first basis for discerning ruffled conformations from planar and other nonplanar structures in ferrous heme proteins.

Electrostatic control of the isoalloxazine environment in the two-electron reduced states of yeast glutathione reductase.

The resonance Raman spectra of the oxidized and two-electron reduced forms of yeast glutathione reductase are reported. The spectra of the oxidized enzyme indicate a low electron density for the isoalloxazine ring. As far as the two-electron reduced species are concerned, the spectral comparison of the NADPH-reduced enzyme with the glutathione- or dithiothreitol-reduced enzyme shows significant frequency differences for the flavin bands II, III, and VII. The shift of band VII was correlated with a change in steric or electronic interaction of the hydroxyl group of a conserved Tyr with the N(10)-C(10a) portion of the isoalloxazine ring. Upward shifts of bands II and III observed for the glutathione- or dithiothreitol-reduced enzyme indicate both a slight change in isoalloxazine conformation and a hydrogen bond strengthening at the N(1) and/or N(5) site(s). The formation of a mixed disulfide intermediate tends to slightly decrease the frequency of bands II, III, X, XI, and XIV. To account for the different spectral features observed for the NADPH- and glutathione-reduced species, several possibilities have been examined. In particular, we propose a hydrogen bonding modulation at the N(5) site of FAD through a variable conformation of an ammonium group of a conserved Lys residue. Changes in N(5)(flavin)-protein interaction in the two-electron reduced forms of glutathione reductase are discussed in relation to a plausible mechanism of the regulation of the enzyme activity via a variable redox potential of FAD.