Alain Desbois

Leghemoglobin green derivatives with nitrated hemes evidence production of highly reactive nitrogen species during aging of legume nodules

Globins constitute a superfamily of proteins widespread in all kingdoms of life, where they fulfill multiple functions, such as efficient O2 transport and modulation of nitric oxide bioactivity. In plants, the most abundant Hbs are the symbiotic leghemoglobins (Lbs) that scavenge O2 and facilitate its diffusion to the N2-fixing bacteroids in nodules. The biosynthesis of Lbs during nodule formation has been studied in detail, whereas little is known about the green derivatives of Lbs generated during nodule senescence. Here we characterize modified forms of Lbs, termed Lbam, Lbcm, and Lbdm, of soybean nodules. These green Lbs have identical globins to the parent red Lbs but their hemes are nitrated. By combining UV-visible, MS, NMR, and resonance Raman spectroscopies with reconstitution experiments of the apoprotein with protoheme or mesoheme, we show that the nitro group is on the 4-vinyl. In vitro nitration of Lba with excess nitrite produced several isomers of nitrated heme, one of which is identical to those found in vivo. The use of antioxidants, metal chelators, and heme ligands reveals that nitration is contingent upon the binding of nitrite to heme Fe, and that the reactive nitrogen species involved derives from nitrous acid and is most probably the nitronium cation. The identification of these green Lbs provides conclusive evidence that highly oxidizing and nitrating species are produced in nodules leading to nitrosative stress. These findings are consistent with a previous report showing that the modified Lbs are more abundant in senescing nodules and have aberrant O2 binding.

Resonance raman spectroscopy of protoheme-protein interactions in oxygen-carrying hemoproteins and in peroxidases

Abstract Resonance Raman spectra of deoxygenated ferrous forms of horse myoglobin, soybean leghemoglobin (isoenzyme a ), monomeric hemoglobin from Glycera, Aplysia myoglobin, horseradish peroxidase (isoenzyme C) and of turnip peroxidase (isoenzymes 1 and 7), excited at 441.6 nm, are reported. Differences observed among these spectra show that the proteins of these two classes of hemoprotein (oxygen-carriers and peroxidases) impose two distinct heme structures which are likely related to their biological functions. Raman bands involving stretching or deformation modes of the vinyl or propionyl groups of the porphyrin occur at different frequencies in the two classes of hemoprotein, reflecting protein-induced differences in conformations of these side-chains. Moreover, resonance Raman spectra allow a diagnosis of vinyl conformations in hemoproteins. More generally, differences in protein environments of the peripheral grous of protoheme in oxygen-carrying hemoproteins and in peroxidases explain the better electron-withdrawing capabilities of these groups in peroxidases than in oxygen-carriers. In addition, the low-frequency regions of resonance Raman spectra show that the axial histidylimidazole is probably deprotonated in the plant peroxidases, while it remains protonated for the oxygen-carriers. This spectroscopic approach, pointing to differences in the peripheral and axial parts of proteheme in the two classes of hemoprotein, provides a new understanding of structure-function relationships of protoheme in hemoproteins.

Low-frequency vibrations of ferroprotoporphyrin-substituted imidazole complexes. A resonance Raman study

This article reports the low-frequency regions of resonance Raman spectra of five- and six-coordinated ferroprotoporphyrin complexes in aqueous solution with or without detergent. For high-spin complexes having their iron atom monocoordinated to variously substituted imidazoles or to dimethylformamide, the frequency of a band observed between 194 and 237 cm−1 (labelled band II) primarily depends on the pKa of the axial ligand. In the absence of steric effects from the axial ligand, the lower is the pKa of ligand, the higher the frequency of band II. We previously assigned band II to a mode essentially involving the Fe-N(pyrrole) bonds. The above pKa dependence is readily explained, in the frame of this assignment, in terms of a decrease in the Fe-N(pyrrole) bond strength (and of an increase in bond length) when the basicity of the axial ligand increases. On the other hand, the alternative assignment of band II to a stretching mode of Fe-N(axial ligand) is inconsistent with the observed pKa dependence. As far as hexacoordinated complexes are concerned, specific bands are observed at 203, 194 and 176 cm−1 for imidazole, 1-methylimidazole and pyridine, respectively. These bands are assigned, on the basis of isotopic substitutions, to a summetric stretching mode of the axial ligands [ν(N-Fe-N)]. Band II is observed at 265 cm−1 for these low-spin complexes, a frequency expected from the short Fe-N(pyrrole) bond lengths of nearly planar ferroporphyrins.

Influence of peripheral substituents on the resonance Raman spectra of ferroporphyrin-2-methylimidazole complexes

Abstract This article reports resonance Raman spectra of 2-methylimidazole complexes of ferroprotoporphyrin IX, ferrohematoporphyrin IX, ferrodeuteroporphyrin IX and of ferroetioporphyrin I excited at 441.6 nm. Comparisons between these spectra show that the frequencies of some bands assigned to modes of the porphyrin skeleton are influenced by inductive effects from peripheral subtituents. These comparisons also permit assignment of modes having a major contribution from pyrrole-peripheral group deformations. Finally, some Raman bands are insensitive to electronic or mass effects from peripheral groupings and constitute markers of the macrocycle structure. These three sets of Raman bands permit extraction of precise information on the structure and environment of hemes in hemoproteins.

Low-frequency heme, iron-ligand, and ligand modes of imidazole and imidazolate complexes of iron protoporphyrin and microperoxidase in aqueous solution. An analysis by far-infrared difference spectroscopy.

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.

Resonance Raman spectra of deoxyhemoproteins. Heme structure in relation to dioxygen binding

Abstract The low-frequency regions of the resonance Raman spectra of deoxygenated ferrous forms of soybean leghemoglobin a , horse myoglobin, sperm whale myoglobin, Aplysia myoglobin, stripped normal human hemoglobin (T quaternary form) and of stripped human NESdesArg-hemoglobin (R quaternary form) are reported. Differences observed among these spectra show that the globins of these hemoproteins impose various heme structures. In particular, the variable frequencies of band II (210–224 cm−1) and of band Ib (121–163 cm−1) show that an increase in dioxygen affinity corresponds to a decrease in Fe-N(pyrrole) bond length.

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 N10–C10a 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 N1 and/or N5 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 N5 site of FAD through a variable conformation of an ammonium group of a conserved Lys residue. Changes in N5(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.

Hydrogen bonding to the cysteine ligand of superoxide reductase: acid–base control of the reaction intermediates

Superoxide reductase (SOR) is a non-heme iron metalloenzyme that detoxifies superoxide radical in microorganisms. Its active site consists of an unusual non-heme Fe2+ center in a [His4Cys1] square pyramidal pentacoordination, with the axial cysteine ligand proposed to be an essential feature in catalysis. Two NH peptide groups from isoleucine 118 and histidine 119 establish hydrogen bonds involving the sulfur ligand (Desulfoarculus baarsii SOR numbering). To investigate the catalytic role of these hydrogen bonds, the isoleucine 118 residue of the SOR from Desulfoarculus baarsii was mutated into alanine, aspartate, or serine residues. Resonance Raman spectroscopy showed that the mutations specifically induced an increase of the strength of the Fe3+–S(Cys) and S–Cβ(Cys) bonds as well as a change in conformation of the cysteinyl side chain, which was associated with the alteration of the NH hydrogen bonding involving the sulfur ligand. The effects of the isoleucine mutations on the reactivity of SOR with O2•− were investigated by pulse radiolysis. These studies showed that the mutations induced a specific increase of the pKa of the first reaction intermediate, recently proposed to be an Fe2+–O2•− species. These data were supported by density functional theory calculations conducted on three models of the Fe2+–O2•− intermediate, with one, two, or no hydrogen bonds involving the sulfur ligand. Our results demonstrated that the hydrogen bonds between the NH (peptide) and the cysteine ligand tightly control the rate of protonation of the Fe2+–O2•− reaction intermediate to form an Fe3+–OOH species.

Protoheme conformations in low-spin ferrohemoproteins. Resonance raman spectroscopy

The low-frequency regions of resonance Raman spectra of various low-spin ferrous forms of normal human hemoglobin, soybean leghemoglobin alpha and of horse myoglobin are reported. Differences observed among the spectra of oxygenated and nitrosyl forms of these hemoproteins show that their globins impose various low-spin heme structures. A quantitative correlation between the variable frequency of resonance Raman band II (215-271 cm-1) and the iron atom-heme plane distance was observed for hemoproteins and heme models, either ferrous or ferric, high-spin or low-spin. From this correlation, the iron atom-heme plane distance should be 0.3 A in nitrosyl and oxymyoglobin (band II at 256 cm-1) whereas the iron position should be near to or in the heme plane for nitrosyl and oxy forms of hemoglobin and leghemoglobin (band II between 266 and 273 cm-1). A new method is proposed for monitoring the photodissociation processes in ferrohemoproteins.

Resonance Raman Study of Heme-Histidine-Protein Interactions in Reduced Cytochromes c’

Cytochromes c’ (cyt c’) are heme proteins isolated from photosynthetic, denitrifying, and nitrogen-fixing bacteria. They exhibit redox potentials in the -10- +100 mV range and constitute a particular class among the c-type cytochromes since, at neutral pH, the iron atom is five-coordinated in both the oxidized and reduced states. This coordination stabilizes a high-spin state for the reduced heme and an admixture of high-spin and intermediate-spin states for the oxidized heme (Meyer & Kamen, 1982). The crystallographic structures of cyt c’ from Rhodospirillum molischianum and Rhodospirillum rubrum show a histidine (His) residue bound to the ferriheme (Weber et al., 1981; Yasui et al., 1992). Considering the particular properties of heme in cyt c’, the characterization of the His-heme-protein interactions appears very important to elucidate. Recently, a resonance Raman (RR) study on a five-coordinated ferroheme c-octapeptide system (MP8(II)) has characterized a mode involving the Fe-N(His) stretching in the 206–243 cm-1 region (Othman et al., 1993). We have therefore investigated the RR spectra of reduced cyt c’ using similar experimental conditions.