Biomedical subjects

Alessandro Giuffrè

Publications and source records attributed to Alessandro Giuffrè.

16 recordsLinked to original sources

PubMed2006

Kinetics of electron transfer from NADH to the Escherichia coli nitric oxide reductase flavorubredoxin.

Escherichia coli flavorubredoxin (FlRd) belongs to the family of flavodiiron proteins (FDPs), microbial enzymes that are expressed to scavenge nitric oxide (NO) under anaerobic conditions. To degrade NO, FlRd has to be reduced by NADH via the FAD-binding protein flavorubredoxin reductase, thus the kinetics of electron transfer along this pathway was investigated by stopped-flow absorption spectroscopy. We found that NADH, but not NADPH, quickly reduces the FlRd-reductase (k = 5.5 +/- 2.2 x 10(6) M(-1).s(-1) at 5 degrees C), with a limiting rate of 255 +/- 17 s(-1). The reductase in turn quickly reduces the rubredoxin (Rd) center of FlRd, as assessed at 5 degrees C working with the native FlRd enzyme (k = 2.4 +/- 0.1 x 10(6) m(-1).s(-1)) and with its isolated Rd-domain (k approximately 1 x 10(7) M(-1).s(-1)); in both cases the reaction was found to be dependent on pH and ionic strength. In FlRd the fast reduction of the Rd center occurs synchronously with the formation of flavin mononucleotide semiquinone. Our data provide evidence that (a) FlRd-reductase rapidly shuttles electrons between NADH and FlRd, a prerequisite for NO reduction in this detoxification pathway, and (b) the electron accepting site in FlRd, the Rd center, is in very fast redox equilibrium with the flavin mononucleotide.

Electron Transport↗

PubMed2006

Nitric oxide reacts with the ferryl-oxo catalytic intermediate of the CuB-lacking cytochrome bd terminal oxidase.

Cytochrome bd is a bacterial respiratory oxidase carrying three hemes but no copper. We show that nitric oxide (NO) reacts with the intermediate F of cytochrome bd from Azotobacter vinelandii: (i) with a 1:1 stoichiometry, (ii) rapidly (k=1.2 +/- 0.1 x 10(5)M(-1)s(-1) at 20 degrees C), and (iii) yielding the oxidized enzyme with nitrite bound to heme d at the active site. Unexpectedly, the NO reaction mechanism of this catalytic intermediate in the Cu(B)-lacking cytochrome bd appears similar to that of beef heart cytochrome c oxidase, where Cu(B) was proposed to play a key role.

Azotobacter vinelandii↗

PubMed2006

Nitric oxide and the respiratory enzyme.

Available information on the molecular mechanisms by which nitric oxide (NO) controls the activity of the respiratory enzyme (cytochrome-c-oxidase) is reviewed. We report that, depending on absolute electron flux, NO at physiological concentrations reversibly inhibits cytochrome-c-oxidase by two alternative reaction pathways, yielding either a nitrosyl- or a nitrite-heme a3 derivative. We address a number of hypotheses, envisaging physiological and/or pathological effects of the reactions between NO and cytochrome-c-oxidase.

Animals↗

PubMed2005

Neuroglobin, nitric oxide, and oxygen: functional pathways and conformational changes.

Neuroglobin (Ngb) is a globin expressed in the nervous system of humans and other organisms that is involved in the protection of the brain from ischemic damage. Despite considerable interest, however, the in vivo function of Ngb is still a conundrum. In this paper we report a number of kinetic experiments with O2 and NO that we have interpreted on the basis of the 3D structure of Ngb, now available for human and murine metNgb and murine NgbCO. The reaction of reduced deoxyNgb with O2 and NO is slow (t(1/2) approximately 2 s) and ligand concentration-independent, because exogenous ligand binding can only occur upon dissociation of the distal His-64, which is coordinated to the ferrous heme iron. By contrast, NgbO2 reacts very rapidly with NO, yielding metNgb and NO3- by means of a heme-bound peroxynitrite intermediate. Steady-state amperometric experiments show that Ngb is devoid of O2 reductase and NO reductase activities. To achieve this result, we have set up a protocol for efficient reduction of metNgb using a mixture of FMN and NADH under bright illumination. The results are discussed with reference to a global scheme inspired by the 3D structures of metNgb and NgbCO. Based on the ligand-linked conformational changes discovered by crystallography, the pathways of the reactions with O2 and NO provide a framework that may account for the involvement of Ngb in controlling the activation of a protective signaling mechanism.

Animals↗

PubMed2005

Nitric oxide, cytochrome c oxidase and myoglobin: competition and reaction pathways.

It is relevant to cell physiology that nitric oxide (NO) reacts with both cytochrome oxidase (CcOX) and oxygenated myoglobin (MbO(2)). In this respect, it has been proposed [Pearce, L.L., et al. (2002) J. Biol. Chem. 277, 13556-13562] that (i) CcOX in turnover out-competes MbO(2) for NO, and (ii) NO bound to reduced CcOX is "metabolized" in the active site to nitrite by reacting with O(2). In contrast, rapid kinetics experiments reported in this study show that (i) upon mixing NO with MbO(2) and CcOX in turnover, MbO(2) out-competes the oxidase for NO and (ii) after mixing nitrosylated CcOX with O(2) in the presence of MbO(2), NO (and not nitrite) dissociates from the enzyme causing myoglobin oxidation.

Animals↗

PubMed2005

Cytochrome c oxidase, ligands and electrons.

We present hereby an overview of the reactions of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, with ligands (primarily oxygen) and electrons, pointing out where necessary unresolved facts or questionable interpretations.

Animals↗

PubMed2005

Probing the access of protons to the K pathway in the Paracoccus denitrificans cytochrome c oxidase.

In recent studies on heme-copper oxidases a particular glutamate residue in subunit II has been suggested to constitute the entry point of the so-called K pathway. In contrast, mutations of this residue (E78(II)) in the Paracoccus denitrificans cytochrome c oxidase do not affect its catalytic activity at all (E78(II)Q) or reduce it to about 50% (E78(II)A); in the latter case, the mutation causes no drastic decrease in heme a(3) reduction kinetics under anaerobic conditions, when compared to typical K pathway mutants. Moreover, both mutant enzymes retain full proton-pumping competence. While oxidized-minus-reduced Fourier-transform infrared difference spectroscopy demonstrates that E78(II) is indeed addressed by the redox state of the enzyme, absence of variations in the spectral range characteristic for protonated aspartic and glutamic acids at approximately 1760 to 1710 cm(-1) excludes the protonation of E78(II) in the course of the redox reaction in the studied pH range, although shifts of vibrational modes at 1570 and 1400 cm(-1) reflect the reorganization of its deprotonated side chain at pH values greater than 4.8. We therefore conclude that protons do not enter the K channel via E78(II) in the Paracoccus enzyme.

Electron Transport Complex IV↗

PubMed2004

Interaction of the bacterial terminal oxidase cytochrome bd with nitric oxide.

Cytochrome bd is a prokaryotic terminal oxidase catalyzing O2 reduction to H2O. The oxygen-reducing site has been proposed to contain two hemes, d and b595, the latter presumably replacing functionally CuB of heme-copper oxidases. We show that NO, in competition with O2, rapidly and potently (Ki = 100 +/- 34 nM at approximately 70 microM O2) inhibits cytochrome bd isolated from Escherichia coli and Azotobacter vinelandii in turnover, inhibition being quickly and fully reverted upon NO depletion. Under anaerobic reducing conditions, neither of the two enzymes reveals NO reductase activity, which is proposed to be associated with CuB in heme-copper oxidases.

Azotobacter vinelandii↗

PubMed2004

Control of cytochrome c oxidase activity by nitric oxide.

Over the past decade it was discovered that, over-and-above multiple regulatory functions, nitric oxide (NO) is responsible for the modulation of cell respiration by inhibiting cytochrome c oxidase (CcOX). As assessed at different integration levels (from the purified enzyme in detergent solution to intact cells), CcOX can react with NO following two alternative reaction pathways, both leading to an effective, fully reversible inhibition of respiration. A crucial finding is that the rate of electron flux through the respiratory chain controls the mechanism of inhibition by NO, leading to either a "nitrosyl" or a "nitrite" derivative. The two mechanisms can be discriminated on the basis of the differential photosensitivity of the inhibited state. Of relevance to cell pathophysiology, the pathway involving the nitrite derivative leads to oxidative degradation of NO, thereby protecting the cell from NO toxicity. The aim of this work is to review the information available on these two mechanisms of inhibition of respiration.

Binding Sites↗

PubMed2004

Proton uptake upon anaerobic reduction of the Paracoccus denitrificans cytochrome c oxidase: a kinetic investigation of the K354M and D124N mutants.

The kinetics and stoichiometry of the redox-linked protonation of the soluble Paracoccus denitrificans cytochrome c oxidase were investigated at pH = 7.2-7.5 by multiwavelength stopped-flow spectroscopy, using the pH indicator phenol red. We compared the wild-type enzyme with the K354M and the D124N subunit I mutants, in which the K- and D-proton-conducting pathways are impaired, respectively. Upon anaerobic reduction by Ru-II hexamine, the wild-type enzyme binds 3.3 +/- 0.6 H(+)/aa(3), i.e., approximately 1 H(+) in excess over beef heart oxidase under similar conditions and the D124N mutant 3.2 +/- 0.5 H(+)/aa(3). In contrast, in the K354M mutant, in which the reduction of heme a(3)-Cu(B) is severely impaired, approximately 0.8 H(+) is promptly bound synchronously with the reduction of heme a, followed by a much slower protonation associated with the retarded reduction of the heme a(3)-Cu(B) site. These results indicate that complete reduction of heme a (and Cu(A)) is coupled to the uptake of approximately 0.8 H(+), which is independent of both H(+)-pathways, whereas the subsequent reduction of the heme a(3)-Cu(B) site is associated with the uptake of approximately 2.5 H(+) transferred (at least partially) through the K-pathway. On the basis of these results, the possible involvement of the D-pathway in the redox-linked protonation of cytochrome c oxidase is discussed.

Amino Acid Substitution↗

PubMed2003

Nitric oxide and cytochrome oxidase: reaction mechanisms from the enzyme to the cell.

The aim of this work is to review the information available on the molecular mechanisms by which the NO radical reversibly downregulates the function of cytochrome c oxidase (CcOX). The mechanisms of the reactions with NO elucidated over the past few years are described and discussed in the context of the inhibitory effects on the enzyme activity. Two alternative reaction pathways are presented whereby NO reacts with the catalytic intermediates of CcOX populated during turnover. The central idea is that at "cellular" concentrations of NO (</= microM), the redox state of the respiratory chain results in the formation of either the nitrosyl- or the nitrite-derivative of CcOX, with potentially different metabolic implications for the cell. In particular, the role played by CcOX in protecting the cell from excess NO, potentially toxic for mitochondria, is also reviewed highlighting the mechanistic differences between eukaryotes and some prokaryotes.

Animals↗

PubMed2003

The molecular mechanisms by which nitric oxide controls mitochondrial complex IV.

This mini-review of focussed on the information available on the molecular mechanisms by which NO controls the function of mitochondrial cytochrome c oxidase and thereby cell respiration. The reaction mechanisms are described as dissected in vitro and recently confirmed in cell cultures, whereby two reaction pathways have been identified, leading to accumulation of either the [a3(2+)NO]-nitrosyl or the [a3(3+)NO2-]-nitrite derivative of the enzyme. The experimental data and the theoretical computation analysis, supporting the hypothesis that one pathway prevails on the other depending on the electron flow level through the respiratory chain, are discussed. Finally, the patho-physiological implications of the reaction between NO and CcOX have been also outlined.

Animals↗

PubMed2002

Redox-linked protonation of cytochrome c oxidase: the effect of chloride bound to CuB.

The effect of bound Cl- on the redox-linked protonation of soluble beef heart cytochrome c oxidase (CcOX) has been investigated at pH 7.3-7.5 by multiwavelength stopped-flow spectroscopy, using phenol red as the pH indicator in an unbuffered medium. Reduction by Ru-II hexamine of the Cl-bound enzyme is associated with an overall apparent uptake of 1.40 +/- 0.21 H+/aa3, whereas 2.28 +/- 0.36 H+/aa3 is taken upon reduction of the Cl-free enzyme. Bound Cl- has no effect on the extent of H+ uptake coupled to heme a reduction (0.59 +/- 0.06 H+/aa3), but significantly decreases (by approximately 0.9 H+/aa3) the apparent stoichiometry of H+ uptake coupled to heme a3-Cu(B) reduction, by eliminating the net H+ uptake linked to Cu(B) reduction. To account for these results, we propose that, after the transfer of the first electron to the active site, reduction of Cu(B) is associated with Cl- dissociation, addition of a H+, and diffusion into the bulk (with subsequent dissociation) of HCl. In the physiologically competent Cl--free enzyme, an OH- likely bound to oxidized Cu(B) is protonated upon arrival of the first electron, and dissociates as H2O. The relevance of this finding to the understanding of the enzyme mechanism is discussed.

Animals↗

PubMed2002

A novel type of nitric-oxide reductase. Escherichia coli flavorubredoxin.

Escherichia coli flavorubredoxin is a member of the family of the A-type flavoproteins, which are built by two core domains: a metallo-beta-lactamase-like domain, at the N-terminal region, harboring a non-heme di-iron site, and a flavodoxin-like domain, containing one FMN moiety. The enzyme from E. coli has an extra module at the C terminus, containing a rubredoxin-like center. The A-type flavoproteins are widespread among strict and facultative anaerobes, as deduced from the analysis of the complete prokaryotic genomes. In this report we showed that the recombinant enzyme purified from E. coli has nitric-oxide reductase activity with a turnover number of approximately 15 mol of NO.mol enzyme(-1).s(-1), which was well within the range of those determined for the canonical heme b(3)-Fe(B) containing nitric-oxide reductases (e.g. approximately 10-50 mol NO.mol enzyme(-1).s(-1) for the Paracoccus denitrificans NOR). Furthermore, it was shown that the activity was due to the A-type flavoprotein core, as the rubredoxin domain alone exhibited no activity. Thus, a novel family of prokaryotic NO reductases, with a non-heme di-iron site as the catalytic center, was established.

Base Sequence↗

PubMed2002

Nitric oxide reacts with the single-electron reduced active site of cytochrome c oxidase.

The reduction kinetics of the mutants K354M and D124N of the Paracoccus denitrificans cytochrome oxidase (heme aa(3)) by ruthenium hexamine was investigated by stopped-flow spectrophotometry in the absence/presence of NO. Quick heme a reduction precedes the biphasic heme a(3) reduction, which is extremely slow in the K354M mutant (k(1) = 0.09 +/- 0.01 s(-1); k(2) = 0.005 +/- 0.001 s(-1)) but much faster in the D124N aa(3) (k(1) = 21 +/- 6 s(-1); k(2) = 2.2 +/- 0.5 s(-1)). NO causes a very large increase (>100-fold) in the rate constant of heme a(3) reduction in the K354M mutant but only a approximately 5-fold increase in the D124N mutant. The K354M enzyme reacts rapidly with O(2) when fully reduced but is essentially inactive in turnover; thus, it was proposed that impaired reduction of the active site is the cause of activity loss. Since at saturating [NO], heme a(3) reduction is approximately 100-fold faster than the extremely low turnover rate, we conclude that, contrary to O(2), NO can react not only with the two-electron but also with the single-electron reduced active site. This mechanism would account for the efficient inhibition of cytochrome oxidase activity by NO in the wild-type enzyme, both from P. denitrificans and from beef heart. Results also suggest that the H(+)-conducting K pathway, but not the D pathway, controls the kinetics of the single-electron reduction of the active site.

Binding Sites↗

PubMedDate not supplied

Nitric oxide and mitochondrial complex IV.

Micromolar nitric oxide (NO) rapidly (ms) inhibits cytochrome c oxidase in turnover with physiological substrates. Two reaction mechanisms have been identified leading, respectively, to formation of a nitrosyl- [a3(2+) -NO] or a nitrite- [a3(3+) -NO2-] derivative of the enzyme. In the presence of O2, the nitrosyl adduct recovers activity slowly, following NO displacement at k' approximately equal to 0.01 s(-1) (37 degrees C); the recovery of the nitrite adduct is much faster. Relevant to pathophysiology, the enzyme does not degrade NO by following the first mechanism, whereas by following the second one it promotes NO oxidation and disposal as nitrite/nitrate. The reaction between NO and cytochrome c oxidase has been investigated at different integration levels of the enzyme, including the in situ state, such as in mouse liver mitochondria or cultured human SY5Y neuroblastoma cells. The respiratory chain is inhibited by NO, either supplied exogenously or produced endogenously via the NO synthase activation. Inhibition of respiration is reversible, although it remains to be clarified whether reversibility is always full and how it depends on concentration of and time of exposure to NO. Oxygraphic measurements show that cultured cells or isolated state 4 mitochondria exposed to micromolar (or less) NO recover from NO inhibition rapidly, as if the nitrite reaction was predominant. Mitochondria in state 3 display a slightly more persistent inhibition than in state 4, possibly due to a higher accumulation of the nitrosyl adduct. Among a number of parameters that appear to control the switch over between the two mechanisms, the concentration of reductants (reduced cytochrome c) at the cytochrome c oxidase site has been proved to be the most relevant one.

Animals↗