Pierre Moënne-Loccoz

Heme oxygenase-1, intermediates in verdoheme formation and the requirement for reduction equivalents.

Conversion of heme to verdoheme by heme oxygenase-1 (HO-1) is thought to involve α-meso-hydroxylation and elimination of the meso-carbon as CO, a reaction supported by both H2O2 and NADPH-cytochrome P450 reductase/O2. Anaerobic reaction of the heme-HO-1 complex with 1 eq of H2O2 produces an enzyme-bound intermediate identified by spectroscopic methods as α-meso-hydroxyheme. This is the first direct evidence for HO-1-catalyzed formation of α-meso-hydroxyheme. α-meso-Hydroxyheme exists as a mixture of Fe(III) phenolate, Fe(III) keto anion, and Fe(II) keto π neutral radical resonance structures. EPR shows that complexation with CO enhances the Fe(II) π neutral radical component. Reaction of the α-meso-hydroxyheme-HO-1 complex with O2 generates Fe(III) verdoheme, which can be reduced in the presence of CO to the Fe(II) verdoheme-CO complex. Thus, conversion of α-meso-hydroxyheme to Fe(III) verdoheme, in contrast to a previous report (Matera, K. M., Takahashi, S., Fujii, H., Zhou, H., Ishikawa, K., Yoshimura, T., Rousseau, D. L., Yoshida, T., and Ikeda-Saito, M. (1996) J. Biol. Chem. 271, 6618-6624), does not require a reducing equivalent. An electron is only required to reduce ferric to ferrous verdoheme in the first step of its conversion to biliverdin.

Transcription factor NsrR from Bacillus subtilis senses nitric oxide with a 4Fe-4S cluster

In Bacillus subtilis, NsrR is required for the upregulation of ResDE-dependent genes in the presence of nitric oxide (NO). NsrR was shown to bind to the promoters of these genes and inhibit their transcription in vitro. NO relieves this inhibition by an unknown mechanism. Here, we use spectroscopic techniques (UV-vis, resonance Raman, and EPR) to show that anaerobically isolated NsrR from B. subtilis contains a [4Fe-4S](2+) cluster, which reacts with NO to form dinitrosyl iron complexes. This method of NO sensing is analogous to that of the FNR protein of Escherichia coli. The Fe-S cluster of NsrR is also reactive toward other exogenous ligands such as cyanide, dithiothreitol, and O(2). These results, together with the fact that there are only three cysteine residues in NsrR, suggest that the 4Fe-4S cluster contains a noncysteinyl labile ligand to one of the iron atoms, leading to high reactivity. Size exclusion chromatography and cross-linking experiments show that NsrR adopts a dimeric structure in its [4Fe-4S](2+) holo form as well as in the apo form. These findings provide a first stepping stone to investigate the mechanism of NO sensing in NsrR.

Secondary coordination sphere influence on the reactivity of nonheme iron(II) complexes: an experimental and DFT approach.

The new biomimetic ligands N4Py2Ph (1) and N4Py2Ph,amide (2) were synthesized and yield the iron(II) complexes [FeII(N4Py2Ph)(NCCH3)](BF4)2 (3) and [FeII(N4Py2Ph,amide)](BF4)2 (5). Controlled orientation of the Ph substituents in 3 leads to facile triplet spin reactivity for a putative FeIV(O) intermediate, resulting in rapid arene hydroxylation. Addition of a peripheral amide substituent within hydrogen-bond distance of the iron first coordination sphere leads to stabilization of a high-spin FeIIIOOR species which decays without arene hydroxylation. These results provide new insights regarding the impact of secondary coordination sphere effects at nonheme iron centers.

Superoxo, μ-peroxo, and μ-oxo complexes from heme/O2 and heme-Cu/O2 reactivity: Copper ligand influences in cytochrome c oxidase models

The O2-reaction chemistry of 1:1 mixtures of (F8)FeII (1; F8 = tetrakis(2,6-diflurorophenyl)porphyrinate) and [(LMe2N)CuI]+ (2; LMe2N = N,N-bis{2-[2-(N′,N′-4-dimethylamino)pyridyl]ethyl}methylamine) is described, to model aspects of the chemistry occurring in cytochrome c oxidase. Spectroscopic investigations, along with stopped-flow kinetics, reveal that low-temperature oxygenation of 1/2 leads to rapid formation of a heme-superoxo species (F8)FeIII-(O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document}) (3), whether or not 2 is present. Complex 3 subsequently reacts with 2 to form [(F8)FeIII–(O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{2-}}}\end{equation*}\end{document})–CuII(LMe2N)]+ (4), which thermally converts to [(F8)FeIII–(O)–CuII(LMe2N)]+ (5), which has an unusually bent (Fe–O–Cu) bond moiety. Tridentate chelation, compared with tetradentate, is shown to dramatically lower the ν(O–O) values observed in 4 and give rise to the novel structural features in 5.

Rational Tuning of the Thiolate Donor in Model Complexes of Superoxide Reductase: Direct Evidence for a Trans Influence in FeIII-OOR Complexes

Iron peroxide species have been identified as important intermediates in a number of nonheme iron as well as heme-containing enzymes, yet there are only a few examples of such species either synthetic or biological that have been well characterized. We describe the synthesis and structural characterization of a new series of five-coordinate (N4S(thiolate))Fe(II) complexes that react with tert-butyl hydroperoxide ((t)BuOOH) or cumenyl hydroperoxide (CmOOH) to give metastable alkylperoxo-iron(III) species (N4S(thiolate)Fe(III)-OOR) at low temperature. These complexes were designed specifically to mimic the nonheme iron active site of superoxide reductase, which contains a five-coordinate iron(II) center bound by one Cys and four His residues in the active form of the protein. The structures of the Fe(II) complexes are analyzed by X-ray crystallography, and their electrochemical properties are assessed by cyclic voltammetry. For the Fe(III)-OOR species, low-temperature UV-vis spectra reveal intense peaks between 500-550 nm that are typical of peroxide to iron(III) ligand-to-metal charge-transfer (LMCT) transitions, and EPR spectroscopy shows that these alkylperoxo species are all low-spin iron(III) complexes. Identification of the vibrational modes of the Fe(III)-OOR unit comes from resonance Raman (RR) spectroscopy, which shows nu(Fe-O) modes between 600-635 cm(-1) and nu(O-O) bands near 800 cm(-1). These Fe-O stretching frequencies are significantly lower than those found in other low-spin Fe(III)-OOR complexes. Trends in the data conclusively show that this weakening of the Fe-O bond arises from a trans influence of the thiolate donor, and density functional theory (DFT) calculations support these findings. These results suggest a role for the cysteine ligand in SOR, and are discussed in light of the recent assessments of the function of the cysteine ligand in this enzyme.

Kinetic and Spectroscopic Studies of Hemin Acquisition in the Hemophore HasAp from Pseudomonas aeruginosa

The extreme limitation of free iron has driven various pathogens to acquire iron from the host in the form of heme. Specifically, several Gram-negative pathogens secrete a heme binding protein known as HasA to scavenge heme from the extracellular environment and to transfer it to the receptor protein HasR for import into the bacterial cell. Structures of heme-bound and apo-HasA homologues show that the heme iron(III) ligands, His32 and Tyr75, reside on loops extending from the core of the protein and that a significant conformational change must occur at the His32 loop upon heme binding. Here, we investigate the kinetics of heme acquisition by HasA from Pseudomonas aeruginosa (HasAp). The rate of heme acquisition from human met-hemoglobin (met-Hb) closely matches that of heme dissociation which suggests a passive mode of heme uptake from this source. The binding of free hemin is characterized by an initial rapid phase forming an intermediate before further conversion to the final complex. Analysis of this same reaction using an H32A variant lacking the His heme ligand shows only the rapid phase to form a heme-protein complex spectroscopically equivalent to that of the wild-type intermediate. Further characterization of these reactions using electron paramagnetic resonance and resonance Raman spectroscopy of rapid freeze quench samples provides support for a model in which heme is initially bound by the Tyr75 to form a high-spin heme-protein complex before slower coordination of the His32 ligand upon closing of the His loop over the heme. The slow rate of this loop closure implies that the induced-fit mechanism of heme uptake in HasAp is not based on a rapid sampling of the H32 loop between open and closed configurations but, rather, that the H32 loop motions are triggered by the formation of the high-spin heme-HasAp intermediate complex.

The millisecond intermediate in the reaction of nitric oxide with oxymyoglobin is an iron(III)--nitrato complex, not a peroxynitrite.

The dioxygenation of nitric oxide by oxyheme in globin proteins is a major route for NO detoxification in aerobic biological systems. In myoglobin, this reaction is thought to proceed through an iron(III)-bound peroxynitrite before homolytic cleavage of the O-O bond to form an iron(IV)-oxo and NO(2) radical followed by recombination and nitrate production. Single turnover experiments at alkaline pH have revealed the presence of a millisecond high-spin heme intermediate. It is widely presumed that this species is an iron(III)-peroxynitrite species, but detailed characterization of the intermediate is lacking. Using resonance Raman spectroscopy and rapid-freeze quench techniques, we identify the millisecond intermediate as an iron(III)-nitrato complex with a symmetric NO(2) stretch at 1282 cm(-1). Greater time resolution techniques will be required to detect the putative iron(III) peroxynitrite complex.

Carboxylate as the Protonation Site in (Peroxo)diiron(III) Model Complexes of Soluble Methane Monooxygenase and Related Diiron Proteins

Addition of H(+) to a synthetic (mu-1,2-peroxo)diiron(III) model complex results in protonation of a carboxylate rather than the peroxo ligand. This conclusion is based on spectroscopic evidence from UV-vis, (57)Fe Mossbauer, resonance Raman, infrared, and (1)H/(19)F NMR studies. These results suggest a similar role for protons in the dioxygen activation reactions in soluble methane monooxygenase and related carboxylate-bridged diiron enzymes.

Phenol Nitration Induced by an {Fe(NO)2}10 Dinitrosyl Iron Complex

Cellular dinitrosyl iron complexes (DNICs) have long been considered NO carriers. Although other physiological roles of DNICs have been postulated, their chemical functionality outside of NO transfer has not been demonstrated thus far. Here we report the unprecedented dioxygen reactivity of a N-bound {Fe(NO)(2)}(10) DNIC, [Fe(TMEDA)(NO)(2)] (1). In the presence of O(2), 1 becomes a nitrating agent that converts 2,4,-di-tert-butylphenol to 2,4-di-tert-butyl-6-nitrophenol via formation of a putative iron-peroxynitrite [Fe(TMEDA)(NO)(ONOO)] (2) that is stable below -80 °C. Iron K-edge X-ray absorption spectroscopy on 2 supports a five-coordinated metal center with a bound peroxynitrite in a cyclic bidentate fashion. The peroxynitrite ligand of 2 readily decays at increased temperature or under illumination. These results suggest that DNICs could have multiple physiological or deleterious roles, including that of cellular nitrating agents.

Influence of the Nitrogen Donors on Nonheme Iron Models of Superoxide Reductase: High-Spin FeIII−OOR Complexes

A new five-coordinate, (N(4)S(thiolate))Fe(II) complex, containing tertiary amine donors, [Fe(II)(Me(4)[15]aneN(4))(SPh)]BPh(4) (2), was synthesized and structurally characterized as a model of the reduced active site of superoxide reductase (SOR). Reaction of 2 with tert-butyl hydroperoxide (tBuOOH) at -78 degrees C led to the generation of the alkylperoxo-iron(III) complex [Fe(III)(Me(4)[15]aneN(4))(SPh)(OOtBu)](+) (2a). The nonthiolate-ligated complex, [Fe(II)(Me(4)[15]aneN(4))(OTf)(2)] (3), was also reacted with tBuOOH and yielded the corresponding alkylperoxo complex [Fe(III)(Me(4)[15]aneN(4))(OTf)(OOtBu)](+) (3a) at an elevated temperature of -23 degrees C. These species were characterized by low-temperature UV-vis, EPR, and resonance Raman spectroscopies. Complexes 2a and 3a exhibit distinctly different spectroscopic signatures than the analogous alkylperoxo complexes [Fe(III)([15]aneN(4))(SAr)(OOR)](+), which contain secondary amine donors. Importantly, alkylation at nitrogen leads to a change from low-spin (S = 1/2) to high-spin (S = 5/2) of the iron(III) center. The resonance Raman data reveal that this change in spin state has a large effect on the nu(Fe-O) and nu(O-O) vibrations, and a comparison between 2a and the nonthiolate-ligated complex 3a shows that axial ligation has an additional significant impact on these vibrations. To our knowledge this study is the first in which the influence of a ligand trans to a peroxo moiety has been evaluated for a structurally equivalent pair of high-spin/low-spin peroxo-iron(III) complexes. The implications of spin state and thiolate ligation are discussed with regard to the functioning of SOR.