Tony A. Mattioli

Site-specific mutagenesis of the reaction centre from Rhodobacter sphaeroides studied by Fourier transform Raman spectroscopy: mutations at tyrosine M210 do not affect the electronic structure of the primary donor

The effects of mutation of residue tyrosine M210 on the primary donor bacteriochlorophylls have been investigated by near infrared FT‐Raman spectroscopy in reaction centres purified from an antenna‐deficient strain of Rhodobacter sphaeroides. We find that mutation at the M210 position does not significantly perturb the distribution of the unpaired electron over the pair of bacteriochlorophyll molecules which constitute the primary donor radical cation. We conclude, therefore, that the effects of mutation of tyrosine M210 on the rate and asymmetry of primary electron transfer in reaction centres cannot be ascribed to a change in the electronic structure of the primary donor.

Identification of iron(III) peroxo species in the active site of the superoxide reductase SOR from Desulfoarculus baarsii.

The active site of superoxide reductase SOR consists of an Fe2+ center in an unusual [His4 Cys1] square-pyramidal geometry. It specifically reduces superoxide to produce H2O2. Here, we have reacted the SOR from Desulfoarculus baarsii directly with H2O2. We have found that its active site can transiently stabilize an Fe3+-peroxo species that we have spectroscopically characterized by resonance Raman. The mutation of the strictly conserved Glu47 into alanine results in a stabilization of this Fe3+-peroxo species, when compared to the wild-type form. These data support the hypothesis that the reaction of SOR proceeds through such a Fe3+-peroxo intermediate. This also suggests that Glu47 might serve to help H2O2 release during the reaction with superoxide.

Application of near-IR Fourier transform resonance Raman spectroscopy to the study of photosynthetic proteins

Abstract We demonstrate the application of near-infrared (NIR) Fourier transform (FT) Raman spectroscopy using 1064 nm excitation to obtain high quality preresonance Raman spectra of bacteriochlorophyll chromophores in photosynthetic proteins from purple bacteria at room temperature without sample degradation. We present NIR FT preresonance Raman spectra of chromatophores from Rhodospirillum (Rsp.) rubrum, carotenoidless strain G9 containing bacteriochlorophyll a (BChl a) chromophores mostly from its B880 antenna complex, and the B850-800 antenna complex from Rhodobacter (Rb.) sphaeroides, 2.4.1 strain; these spectra are compared with their resonance Raman (RR) spectra obtained using 363.8 nm excitation at 30 K. For antenna complexes not containing carotenoid the FT Raman spectra are dominated by the vibrational modes of the bacteriochlorophyll chromophores with no interference from the modes of the protein or membrane. In the NIR FT Raman spectrum of the B850-800 complex from Rb. sphaeroides 2.4.1, strong contributions from the carotenoid molecule are observed to cause some interference with the 1609 cm−1 band of the BChl a molecules. We also present FT Raman spectra of reaction centers (RCs) from Rb. sphaeroides R 26 in the reduced and oxidized states of their primary electron donor (P). In the reduced state, it is estimated that ca 70% of the FT Raman spectrum arises from reduced P whose electronic absorption band is at 865 nm. With 1064 nm excitation of the RCs poised in the oxidized cation radical state of P, we have observed for the first time a Raman spectrum of P+. via resonance in the vibronic region of the 1250 nm absorption band of this species. This spectrum indicates that the unpaired electron in dimeric P+. is not equally shared between the two BChl molecules constituting the primary donor. Spectral contributions of the carotenoid in the FT RR spectra of wild-type Rb. sphaeroides (2.4.1) RCs confirm it is assuming an out-of-plane distorted, 15-cis configuration under the specific conditions offered by FT Raman spectroscopy, i.e. with the sample at room temperature and using an excitation wavelength which is non-actinic for the carotenoid.

Activation of peroxynitrite by inducible nitric-oxide synthase: a direct source of nitrative stress.

In mammals, nitric oxide (NO) is an essential biological mediator that is exclusively synthesized by nitric-oxide synthases (NOSs). However, NOSs are also directly or indirectly responsible for the production of peroxynitrite, a well known cytotoxic agent involved in numerous pathophysiological processes. Peroxynitrite reactivity is extremely intricate and highly depends on activators such as hemoproteins. NOSs present, therefore, the unique ability to both produce and activate peroxynitrite, which confers upon them a major role in the control of peroxynitrite bioactivity. We report here the first kinetic analysis of the interaction between peroxynitrite and the oxygenase domain of inducible NOS (iNOSoxy). iNOSoxy binds peroxynitrite and accelerates its decomposition with a second order rate constant of 22 × 104 m–1s–1 at pH 7.4. This reaction is pH-dependent and is abolished by the binding of substrate or product. Peroxynitrite activation is correlated with the observation of a new iNOS heme intermediate with specific absorption at 445 nm. iNOSoxy modifies peroxynitrite reactivity and directs it toward one-electron processes such as nitration or one-electron oxidation. Taken together our results suggest that, upon binding to iNOSoxy, peroxynitrite undergoes homolytic cleavage with build-up of an oxo-ferryl intermediate and concomitant release of a \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{2}^{{\cdot}}\) \end{document} radical. Successive cycles of peroxynitrite activation were shown to lead to iNOSoxy autocatalytic nitration and inhibition. The balance between peroxynitrite activation and self-inhibition of iNOSoxy may determine the contribution of NOSs to cellular oxidative stress.

Role of Arginine Guanidinium Moiety in Nitric-oxide Synthase Mechanism of Oxygen Activation

Nitric-oxide synthases (NOS) are highly regulated heme-thiolate enzymes that catalyze two oxidation reactions that sequentially convert the substrate l-Arg first to Nω-hydroxyl-l-arginine and then to l-citrulline and nitric oxide. Despite numerous investigations, the detailed molecular mechanism of NOS remains elusive and debatable. Much of the dispute in the various proposed mechanisms resides in the uncertainty concerning the number and sources of proton transfers. Although specific protonation events are key features in determining the specificity and efficiency of the two catalytic steps, little is known about the role and properties of protons from the substrate, cofactors, and H-bond network in the vicinity of the heme active site. In this study, we have investigated the role of the acidic proton from the l-Arg guanidinium moiety on the stability and reactivity of the ferrous heme-oxy complex intermediate by exploiting a series of l-Arg analogues exhibiting a wide range of guanidinium pKa values. Using electrochemical and vibrational spectroscopic techniques, we have analyzed the effects of the analogues on the heme, including characteristics of its proximal ligand, heme conformation, redox potential, and electrostatic properties of its distal environment. Our results indicate that the substrate guanidinium pKa value significantly affects the H-bond network near the heme distal pocket. Our results lead us to propose a new structural model where the properties of the guanidinium moiety finely control the proton transfer events in NOS and tune its oxidative chemistry. This model may account for the discrepancies found in previously proposed mechanisms of NOS oxidation processes.

Folding of the prion peptide GGGTHSQW around the copper(II) ion: identifying the oxygen donor ligand at neutral pH and probing the proximity of the tryptophan residue to the copper ion.

The GGGTHSQW sequence in the amyloidogenic part of the prion protein is a potential binding site for Cu(II). We have previously studied the binding of copper to the shorter GGGTH peptide and showed that it is highly pH dependent (Hureau et al. in J. Biol. Inorg. Chem. 11:735–744, 2006). Two predominant complexes could be characterized at pH 6.7 and 9.0 with equatorial binding modes of 3N1O and 4N for the metal ion, respectively. In this work, we have further investigated the coordination of Cu(II) to the GGGTH peptide as well as the longer GGGTHSQW peptide in order to identify the oxygen donor ligand at neutral pH and to study the proximity and redox activity of the tryptophan residue of the latter. The results for both peptides show that, at pH 6.7, Cu(II) is coordinated by a carbonyl peptide backbone. At higher pH values, the carbonyl ligand dissociates and the coordination changes to a 4N binding mode, inducing a structural rearrangement that brings the GGGTHSQW peptide’s tryptophan residue into the vicinity of the copper ion, thus affecting their respective redox properties.

Assessing the Role of the Active-site Cysteine Ligand in the Superoxide Reductase from Desulfoarculus baarsii

Superoxide reductase is a novel class of non-heme iron proteins that catalyzes the one-electron reduction of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{.}}}\) \end{document} to H2O2, providing an antioxidant defense in some bacteria. Its active site consists of an unusual non-heme Fe2+ center in a [His4 Cys1] square pyramidal pentacoordination. In this class of enzyme, the cysteine axial ligand has been hypothesized to be an essential feature in the reactivity of the enzyme. Previous Fourier transform infrared spectroscopy studies on the enzyme from Desulfoarculus baarsii revealed that a protonated carboxylate group, proposed to be the side chain of Glu114, is in interaction with the cysteine ligand. In this work, using pulse radiolysis, Fourier transform infrared, and resonance Raman spectroscopies, we have investigated to what extent the presence of this Glu114 carboxylic lateral chain affects the strength of the S—Fe bond and the reaction of the iron active site with superoxide. The E114A mutant shows significantly modified pulse radiolysis kinetics for the protonation process of the first reaction intermediate. Resonance Raman spectroscopy demonstrates that the E114A mutation results in both a strengthening of the S—Fe bond and an increase in the extent of freeze-trapping of a Fe-peroxo species after treatment with H2O2 by a specific strengthening of the Fe—O bond. A fine tuning of the strength of the S—Fe bond by the presence of Glu114 appears to be an essential factor for both the strength of the Fe—O bond and the pKa value of the Fe3+-peroxo intermediate species to form the reaction product H2O2.

The influence of hydrogen bonds on the electronic structure of light-harvesting complexes from photosynthetic bacteria.

The influence of hydrogen bonds on the electronic structure of the light-harvesting I complex from Rhodobacter sphaeroides has been examined by site-directed mutagenesis, steady-state optical spectroscopy, and Fourier-transform resonance Raman spectroscopy. Shifts of 4-23 nm in the Q(y) absorption band were observed in seven mutants with single or double changes at Leu alpha44, Trp alpha43, and Trp beta48. Resonance Raman spectra were consistent with the loss of a hydrogen bond with the alteration of either Trp alpha43 or Trp beta48 to Phe. However, when the Trp alpha43 to Phe alteration is combined with Leu alpha44 to Tyr, the spectra show that the loss of the hydrogen bond to alpha43 is compensated by the addition of a new hydrogen bond to Tyr alpha44. Comparison of the absorption and vibrational spectra of the seven mutants suggests that changes in the absorption spectra can be interpreted as being due to both structural and hydrogen-bonding changes. To model these changes, the structural and hydrogen bond changes are considered to be independent of each other. The calculated shifts agree within 1 nm of the observed values. Excellent agreement is also found assuming that the structural changes arise from rotations of the C3-acetyl group conformation and hydrogen bonding. These results provide the basis for a simple model that describes the effect of hydrogen bonds on the electronic structures of the wild-type and mutant light-harvesting I complexes and also is applicable for the light-harvesting II and light-harvesting III complexes. Other possible effects of the mutations, such as changes in the disorder of the environment of the bacteriochlorophylls, are discussed.

A COMPARATIVE STUDY OF CONSERVED PROTEIN INTERACTIONS OF THE PRIMARY ELECTRON DONOR IN PHOTOSYNTHETIC PURPLE BACTERIAL REACTION CENTERS

The pigment–protein interactions within the binding site of the bacteriochlorophyll (BChl) dimer constituting the primary electron donor (P) in several, native, photosynthetic bacterial reaction centers have been determined using Fourier transform Raman spectroscopy. For reaction centers whose primary sequence data are available, and assuming a structural analogy with the Rb. sphaeroides RC whose high-resolution three-dimensional structure is known, amino acid residues donating hydrogen bonds to P are proposed. Consequently, one may propose the microenvironment structure of the primary donors studied and correlate this deduced structure with the known absorption and redox properties of the primary donors. In this ‘mini-review’ of our past work, we group and classify the primary donors with respect to their specific H-bonding interactions with the protein. This classification reveals trends in the H-bonding and certain physicochemical properties such as the P°/P⋅+ redox midpoint potential, the positive charge distribution over the dimeric primary donors in their oxidized radical cation state P⋅+, and the absorption maxima of the lower exciton Qy absorption band of P.

NO synthase isoforms specifically modify peroxynitrite reactivity

Nitric oxide synthases (NOSs) are multi‐domain hemothiolate proteins that are the sole source of nitric oxide (NO) in mammals. NOSs can also be a source or a sink for peroxynitrite (PN), an oxidant that is suspected to be involved in numerous physiopathological processes. In a previous study, we showed that the oxygenase domain of the inducible NOS (iNOSoxy) reacts with PN and changes its oxidative reactivity [Maréchal A, Mattioli TA, Stuehr DJ & Santolini J (2007) J Biol Chem282, 14101–14112]. Here we report a similar analysis on two other NOS isoforms, neuronal NOS (nNOS) and a bacterial NOS‐like protein (bsNOS). All NOSs accelerated PN decomposition, with accumulation of a similar heme intermediate. The kinetics of PN decomposition and heme transitions were comparable among NOSs. However, their effects on PN reactivity differ greatly. All isoforms suppressed PN two‐electron oxidative activity, but iNOSoxy enhanced PN one‐electron oxidation and nitration potencies, the oxygenase domain of nNOS (nNOSoxy) affected them minimally, and bsNOS abolished all PN reactivities. This led to the loss of both NOS and PN decomposition activities for nNOSoxy and iNOSoxy, which may be linked to the reported alterations in their electronic absorption spectra. Bacterial bsNOS was affected to a lesser extent by reaction with PN. We propose that these differences in PN reactivity among NOSs might arise from subtle differences in their heme pockets, and could reflect the physiological specificity of each NOS isoform, ranging from oxidative stress amplification (iNOS) to detoxification (bsNOS).