PubMed Health⌕ Search

Biomedical subjects

D E Edmondson

Publications and source records attributed to D E Edmondson.

At least 19 recordsLinked to original sources
PubMed2006

Structure of the human mitochondrial monoamine oxidase B: new chemical implications for neuroprotectant drug design.

Monoamine oxidase B (MAO-B) is an outer mitochondrial membrane-bound flavoenzyme that is a well-known target for antidepressant and neuroprotective drugs. The 3A resolution structure of recombinant human MAO-B originally determined was of the enzyme complexed with pargyline, an irreversible inhibitor covalently bound to the N5 atom of the flavin coenzyme. The crystal structure shows that the enzyme is dimeric. Each monomer binds to the membrane via a C-terminal transmembrane helix and by apolar loops located at various positions in the sequence. Substrate binding to the enzyme involves negotiating a loop covering a 290A3 entrance apolar cavity before reaching an apolar 420A3 substrate cavity where the flavin coenzyme is located. The 1.7A isatin-MAO-B structure allowed a detailed examination of the enzyme's active site. A novel specific reversible MAO-B inhibitor, which is found as a contaminant in polystyrene plastics (1,4-diphenyl-2-butene), binds in both the entrance and the substrate cavity. Analogous MAO-B-specific inhibitors that bind in a manner traversing both cavities include trans-trans farnesol and chlorostyrylcaffeine. The rotation of the Ile199 side chain to an "open" conformation plays an essential role in this specificity. These results form a molecular basis for the design of new human MAO-B-specific reversible inhibitors.

Binding, Competitive↗
PubMed2004

Structure and mechanism of monoamine oxidase.

Monoamine oxidases A and B (MAO A and MAO B) are mitochondrial outer membrane-bound flavoproteins that catalyze the oxidative deamination of neurotransmitters and biogenic amines. A number of mechanism-based inhibitors (MAOI's) have been developed for clinical use as antidepressants and as neuroprotective drugs. To facilitate the development of more effective and specific inhibitors, a detailed understanding of the structures and catalytic mechanisms of these enzymes is required. The recent development of high level expression systems for producing recombinant human liver MAO A and MAO B in Pichia pastoris has facilitated the determination of the three dimensional crystal structures of MAO B (up to 1.7 angstroms resolution) in complex with different reversible (isatin, 1,4-diphenyl-2-butene) and irreversible inhibitors (pargyline, N-(2-aminoethyl)-p-chlorobenzamide, and trans-2-phenylcyclopropylamine). The binding of substrates or inhibitors to MAO B involves an initial negotiation of a protein loop occurring near the surface of the membrane and two hydrophobic cavities; an "entrance" cavity and an "active site" cavity. These two cavities can either be separate or in a fused state depending on the conformation of the Ile199 side chain, which appears to function as a gate. The amine function of the bound substrate approaches the re face of the bent and "puckered" covalent FAD through an "aromatic cage" formed by two tyrosine residues that are perpendicular to the plane of the flavin ring. No amino acid residues that could function as acids or bases are found near the catalytic site. The existing structural data on MAO B support previous QSAR results and are also supportive of a proposed polar nucleophilic mechanism for MAO A and B catalysis rather than the alternatively proposed single electron transfer mechanism.

Amino Acid Sequence↗
PubMed2001

Loss of serotonin oxidation as a component of the altered substrate specificity in the Y444F mutant of recombinant human liver MAO A.

To investigate the roles of tyrosyl residues located near the covalent 8alpha-S-cysteinyl FAD in monoamine oxidase A (MAO A) and to test the suggestion that MAO A and plant polyamine oxidase may have structural homology, tyrosyl to phenylalanyl mutants of MAO A at positions 377, 402, 407, 410, 419, and 444 were constructed and expressed in Saccharomyces cerevisiae. All mutant enzymes were expressed and exhibited lower specific activities as compared to WT MAO A using kynuramine as substrate. The lowest specific activities in this assay are exhibited by the Y407F and Y444F mutant enzymes. On purification and further characterization, these two mutants were found to each contain covalent FAD. Both mutant enzymes are irreversibly inhibited by the MAO A inhibitor clorgyline and exhibit binding stoichiometries of 0.54 (Y407F) and 0.95 (Y444F) as compared to 1.05 for WT MAO A. Y444F MAO A oxidizes kynuramine with a k(cat) <2% of WT enzyme and is greater than 100-fold slower in catalyzing the oxidation of phenylethylamine or of serotonin. In contrast, Y444F MAO A oxidizes p-CF(3)-benzylamine at a rate 25% that of WT enzyme. Steady state and reductive half-reaction stopped-flow data using a series of para-substituted benzylamine analogues show Y444F MAO A exhibits quantitative structure activity relationships (QSAR) properties on analogue binding and rates of substrate oxidation very similar to that exhibited by the WT enzyme (Miller and Edmondson (1999) Biochemistry 38, 13670): log K(d) = -(0.37 +/- ()()0.07)V(W)(x0.1) - 4.5 +/- 0.1; log k(red) = +(2.43 +/- 0.19)sigma + 0.17 +/- 0.05. The Y444F MAO A mutant also exhibits similar QSAR properties on the binding of phenylalkyl side chain amine analogues as WT enzyme: log K(i) = (4.37 +/- 0.51)E(S) + 1.21 +/- 0.77. These data show that mutation of Y444F in MAO A results in a mutant that has lost its ability to efficiently oxidize serotonin (its physiological substrate) but, however, exhibits unaltered quantitative structure-activity parameters in the binding and rate of benzylamine analogues. The mechanism of C-H abstraction is therefore unaltered. The suggestion that polyamine oxidase and monoamine oxidase may have structural homology appears to be valid as regards Y444 in MAO A and Y439 in plant polyamine oxidase.

Animals↗
PubMed2001

The covalent FAD of monoamine oxidase: structural and functional role and mechanism of the flavinylation reaction.

The family of flavoenzymes in which the flavin coenzyme redox cofactor is covalently attached to the protein through an amino acid side chain is covered in this review. Flavin-protein covalent linkages have been shown to exist through each of five known linkages: (a) 8alpha-N(3)-histidyl, (b) 8alpha-N(1)-histidyl, (c) 8alpha-S-cysteinyl, (d) 8alpha-O-tyrosyl, or (e) 6-S-cysteinyl with the flavin existing at either the flavin mononucleotide or flavin adenine dinucleotide (FAD) levels. This class of enzymes is widely distributed in diverse biological systems and catalyzes a variety of enzymatic reactions. Current knowledge on the mechanism of covalent flavin attachment is discussed based on studies on the 8alpha-S-cysteinylFAD of monoamine oxidases A and B, as well as studies on other flavoenzymes. The evidence supports an autocatalytic quinone-methide mechanism of protein flavinylation. Proposals to explain the structural and mechanistic advantages of a covalent flavin linkage in flavoenzymes are presented. It is concluded that multiple factors are involved and include: (a) stabilization of the apoenzyme structure, (b) steric alignment of the cofactor in the active site to facilitate catalysis, and (c) modulation of the redox potential of the covalent flavin through electronic effects of 8alpha-substitution.

Benzoquinones↗
PubMed2000

Structure-activity relations in the oxidation of phenethylamine analogues by recombinant human liver monoamine oxidase A.

The interaction of recombinant human liver monoamine oxidase A (MAO A) with a series of phenethylamine substrate analogues has been investigated by steady-state and stopped-flow kinetic techniques. Substrate analogues with para substituents exhibit large deuterium kinetic isotope effect on k(cat), on k(cat)/K(m), and on the limiting rate of enzyme reduction in reductive half-reaction experiments. These kinetic isotope effect values range from 5 to 10 with the exception of tyramine, which exhibited smaller steady-state isotope effects (2.3-3.5) than that observed on the rate of flavin reduction (6.9). The stopped-flow data show that imine release from the reduced enzyme is slower than the rate of catalytic turnover. Phenethylamine oxidation by MAO A can be described as the C-H bond cleavage step being rate limiting in catalysis and with oxygen reacting with the reduced enzyme-imine complex. In the case of tyramine, the product release from the oxidized enzyme-imine complex contributes to the rate limitation in catalysis. The binding affinities of a series of para-substituted phenethylamine analogues to MAO A show an increase in affinity of the deprotonated amine with increasing van der Waals volume of the substituent. The limiting rate of enzyme reduction decreases with increasing van der Waals volume of the substituent in a linear manner with no observable electronic contribution as observed previously with benzylamine reduction of MAO A [Miller, J. R., and Edmondson, D. E. (1999) Biochemistry 38, 13670-13683]. Examination of side chain analogues of phenethylamine show 3-phenylpropylamine to be oxidized 2.5-fold more slowly and bound 75-fold more tightly than phenethylamine. 4-Phenylbutylamine is not a substrate for MAO A but is a good competitive inhibitor with a K(i) value of 31 +/- 5 microM. Analysis of the effect of alkyl side chain alterations on binding affinities of a series of arylalkylamine analogues taken from this study and from the literature show a linear correlation with the Taft steric value (E(s)) of the side chain. These results suggest that the binding site for the aryl ring is identical for phenethylamine and for benzylamine analogues and that steric interactions of the alkyl side chain with the enzyme strongly contribute to the binding affinities of a series of reversible inhibitors of MAO A.

Flow Injection Analysis↗
PubMed2000

Influence of FAD structure on its binding and activity with the C406A mutant of recombinant human liver monoamine oxidase A.

The FAD binding site of human liver monoamine oxidase A (MAO A) has been investigated by mutagenesis of the amino acid site of covalent FAD attachment (Cys-406) to an alanyl residue. Expression of the C406A mutant in Saccharomyces cerevisiae results in the formation of an active enzyme, as found previously with the rat liver enzyme. The activity of this mutant enzyme is labile to solubilization, thus requiring all experiments to be done with membrane preparations. C406A MAO A was expressed in a rib 5(-) strain of S. cerevisiae in the presence of 16 different riboflavin analogues. Inactive apoC406A MAO A is formed by induction of the enzyme in the absence of riboflavin. FAD but not FMN or riboflavin restores catalytic activity with an apparent K(d) of 62 +/- 5 nm. The results from both in vivo and in vitro reconstitution experiments show increased activity levels (up to approximately 7-fold higher) with those analogues exhibiting higher oxidation-reduction potentials than normal flavin and decreased activity levels with analogues exhibiting lower potentials. Analogues with substituents on the pyrimidine ring bind to C406A MAO A more weakly than normal FAD, suggesting specific interactions with the N(3) and N(1) positions. Analogues with substituents in the 7 and 8 positions bind to C406A MAO A with affinities comparable with that of normal FAD. These results are discussed in regard to functional significance of 8alpha-covalent binding of flavins to proteins.

Binding Sites↗
PubMed2000

Evidence for alternative binding modes in the interaction of benzylamine analogues with bovine liver monoamine oxidase B.

The interaction of purified bovine liver MAO B with the benzylamine analogues N,N-dimethylbenzylamine and alpha-methylbenzylamine has been investigated. Both classes of analogues are competitive inhibitors of benzylamine oxidase activity. The K(i) values were determined for nine different para-substituted N, N-dimethylbenzylamine analogues. Analysis of the binding affinities demonstrate the deprotonated forms of the tertiary amines are preferentially bound to MAO B and the affinity decreases with increasing van der Waals volume of the para-substituent. The correlation for this relation is:Log K(i)=-0.97+/-(0.28)sigma+(0. 75+/-0.11)(0.1xV(w))-4.24+/-(0.16)alpha-Methyl benzylamine analogues are also found to be competitive inhibitors of MAO B-catalyzed benzylamine oxidation. Similar K(i) values were determined using either the S or R stereoisomers. Analysis of the binding affinities of five para-substituted alpha-methylbenzylamine analogues to MAO B shows the deprotonated form also to be preferentially bound and the affinity is marginally increased with increasing van der Waals volume of the para-substituent:Log K(i)=-0.71sigma-(0.32)(0. 1xV(w))-3.50Comparison of these data with that previously published for para-substituted benzylamine binding to MAO B (Walker and Edmondson, Biochemistry 33 (1994) 7088-7098) demonstrates that these benzylamine analogues exhibit differing modes of binding to the active site of MAO B. The presence of an electronic substituent effect in the binding of these two classes of analogues compared with the lack of an observable electronic effect in the binding of benzylamine to MAO B is consistent with the proposal that orientation of the benzyl ring of the bound substrate is responsible for the absence of an electronic substituent effect on the rate of the reductive half reaction (Miller and Edmondson, Biochemistry 38 (1999) 13670-13683).

Animals↗
PubMed2000

A short Fe-Fe distance in peroxodiferric ferritin: control of Fe substrate versus cofactor decay?

The reaction of oxygen with protein diiron sites is important in bioorganic syntheses and biomineralization. An unusually short Fe-Fe distance of 2.53 angstroms was found in the diiron (mu-1,2 peroxodiferric) intermediate that forms in the early steps of ferritin biomineralization. This distance suggests the presence of a unique triply bridged structure. The Fe-Fe distances in the mu-1, 2 peroxodiferric complexes that were characterized previously are much longer (3.1 to 4.0 angstroms). The 2.53 angstrom Fe-Fe distance requires a small Fe-O-O angle (approximately 106 degrees to 107 degrees). This geometry should favor decay of the peroxodiferric complex by the release of H2O2 and mu-oxo or mu-hydroxo diferric biomineral precursors rather than by oxidation of the organic substrate. Geometrical differences may thus explain how diiron sites can function either as a substrate (in ferritin biomineralization) or as a cofactor (in O2 activation).

Chemical Phenomena↗
PubMed2000

The FAD binding sites of human liver monoamine oxidases A and B: investigation of the role of flavin ribityl side chain hydroxyl groups in the covalent flavinylation reaction and catalytic activities.

The role of ribityl side chain hydroxyl groups of the flavin moiety in the covalent flavinylation reaction and catalytic activities of recombinant human liver monoamine oxidases (MAO) A and B have been investigated using the riboflavin analogue: N(10)-omega-hydroxypentyl-isoalloxazine. Using a rib5 disrupted strain of Saccharomyces cerevisiae which is auxotrophic for riboflavin, MAO A and MAO B were expressed separately under control of a galactose inducible GAL10/CYC1 promoter in the presence of N(10)-omega-hydroxypentyl-isoalloxazine as the only available riboflavin analogue. Analysis of mitochondrial membrane proteins shows both enzymes to be expressed at levels comparable to those cultures grown on riboflavin and to contain covalently bound flavin. Catalytic activities, as monitored by kynuramine oxidation, are equivalent to (MAO A) or 2-fold greater (MAO B) than control preparations expressed in the presence of riboflavin. Although N(10)-omega-hydroxypentyl-isoalloxazine is unable to support growth of riboflavin auxotrophic S. cerevisiae, it is converted to the FMN level by yeast cell free extracts. The FMN form of the analogue is converted to the FAD level by the yeast FAD synthetase, as shown by expression of the recombinant enzyme in Escherichia coli. These data show that the ribityl hydroxyl groups of the FAD moiety are not required for covalent flavinylation or catalytic activities of monoamine oxidases A and B. This is in contrast to the suggestion based on mutagenesis studies that an interaction between the 3'-hydroxyl group of the flavin and the beta-carbonyl of Asp(227) is required for the covalent flavinylation reaction of MAO B (Zhou et al., J. Biol. Chem. 273 (1998) 14862-14868).

Binding Sites↗
PubMed2000

High-level expression of human liver monoamine oxidase B in Pichia pastoris.

The high-level heterologous expression, purification, and characterization of the mitochondrial outer membrane enzyme human liver monoamine oxidase B (MAO B) using the methylotrophic yeast Pichia pastoris expression system are described. A 2-L culture of P. pastoris expresses approximately 1700 U of MAO B activity, with the recombinant enzyme associated tightly with the membrane fraction of the cell lysate. By a modification of the published procedure for purification of bovine liver MAO B [Salach, J. I. (1979) Arch. Biochem. Biophys. 192, 128-137], recombinant human liver MAO B is purified in a 34% yield ( approximately 200 mg from 2 L of cell culture). The isolated enzyme exhibits an M(r) of approximately 60, 000 on SDS-PAGE and 59,474 from electrospray mass spectrometry measurements, which is in good agreement with the mass predicted from the gene sequence and inclusion of the covalent FAD. One mole of covalent FAD per mole of MAO B is present in the purified enzyme and is bound by an 8alpha-S-cysteinyl(397) linkage, as identified by electrospray mass spectrometry of the isolated tryptic/chymotryptic flavin peptide. Recombinant human liver MAO B and bovine liver MAO B are shown to be acetylated at the seryl residues at their respective amino termini. The benzylamine oxidase activity of recombinant MAO B ranges from 3.0 to 3.4 U/mg and steady-state kinetic parameters for this enzyme preparation compare well with those published for the bovine liver enzyme: k(cat) = 600 min(-1), K(m)(benzylamine) = 0.50 mM, and K(m)(O(2)) = 0.33 mM. Kinetic isotope effect parameters using [alpha,alpha-(2)H(2)]benzylamine are also similar to those found for the bovine enzyme. Recombinant MAO B exhibits a (D)k(cat) = 4.7, a (D)[k(cat)/K(m)(benzylamine)] = 4.5, and a (D)[k(cat)/K(m)(O(2))] = 1.0. In contrast to bovine liver MAO B, no evidence was found for the presence of any anionic flavin radical either by UV-vis or by EPR spectroscopy in the resting form of the enzyme. These data demonstrate the successful heterologous expression of a functional, membrane-bound MAO B, which will permit a number of mutagenesis studies as structural and mechanistic probes not previously possible.

Acetylation↗
PubMed1999

Formation of a pterin radical in the reaction of the heme domain of inducible nitric oxide synthase with oxygen.

The heme domain (iNOS(heme)) of inducible nitric oxide synthase (NOS) was expressed in Escherichia coli and purified to homogeneity. Rapid freeze-quench (RFQ) EPR was used to monitor the reaction of the reduced iNOS(heme) with oxygen in the presence and absence of substrate. In these reactions, heme oxidation occurs at a rate of approximately 15 s(-)(1) at 4 degrees C. A transient species with a g = 2.0 EPR signal is also observed under these conditions. The spectral properties of the g = 2.0 signal are those of an anisotropic organic radical with S = (1)/(2). Comparison of the EPR spectra obtained when iNOS(heme) is reconstituted with N5-(14)N- and (15)N-substituted tetrahydrobiopterin (H(4)B) shows a hyperfine interaction with the pterin N5 nitrogen and identifies the radical as the one-electron oxidized form (H(3)B.) of the bound H(4)B. Substitution of D(2)O for H(2)O reveals the presence of hyperfine-coupled exchangeable protons in the H(4)B radical. This radical forms at a rate of 15-20 s(-)(1), with a slower decay rate that varies (0.12-0.7 s(-)(1)) depending on the substrate. At 127 ms, H(3)B. accumulates to a maximum of 80% of the total iNOS(heme) concentration in the presence of arginine but only to approximately 2.8% in the presence of NHA. Double-mixing RFQ experiments, where NHA is added after the formation of H(3)B., show that NHA does not react rapidly with H(3)B. and suggest that NHA instead prevents the formation of the H(4)B radical. These data constitute the first direct evidence for an NOS-bound H(3)B. and are most consistent with a role for H(4)B in electron transfer in the NOS reaction.

Arginine↗
PubMed1999

Structure-activity relationships in the oxidation of para-substituted benzylamine analogues by recombinant human liver monoamine oxidase A.

Monoamine oxidase A (MAO A) plays a central role in the oxidation of amine neurotransmitters. To investigate the structure and mechanism of this enzyme, recombinant human liver MAO A was expressed and purified from Saccharomyces cerevisiae. Anaerobic titrations of the enzyme require only 1 mol of substrate per mole of enzyme-bound flavin for complete reduction. This demonstrates that only one redox-active group (i.e., the covalent FAD cofactor) is involved in catalysis. The reaction rates and binding affinities of 17 para-substituted benzylamine analogues with purified MAO A were determined by steady state and stopped flow kinetic experiments. For each substrate analogue that was tested, the rates of steady state turnover (k(cat)) and anaerobic flavin reduction (k(red)) are similar in value. Deuterium kinetic isotope effects on k(cat), k(red), k(cat)/K(m), and k(red)/K(s) with alpha, alpha-[(2)H]benzylamines are similar for each substrate analogue that was tested and range in value from 6 to 13, indicating that alpha-C-H bond cleavage is rate-limiting in catalysis. Substrate analogue dissociation constants determined from reductive half-reaction experiments as well as from steady state kinetic isotope effect data [Klinman, J. P., and Matthews, R. G. (1985) J. Am. Chem. Soc. 107, 1058-1060] are in excellent agreement. Quantitative structure-activity relationship (QSAR) analysis of dissociation constants shows that the binding of para-substituted benzylamine analogues to MAO A is best correlated with the van der Waals volume of the substituent, with larger substituents binding most tightly. The rate of para-substituted benzylamine analogue oxidation and/or substrate analogue-dependent flavin reduction is best correlated with substituent electronic effects (sigma). Separation of the electronic substituent parameter (sigma) into field-inductive and resonance effects provides a more comprehensive treatment of the electronic correlations. The positive correlation of rate with sigma (rho approximately 2.0) suggests negative charge development at the benzyl carbon position occurs and supports proton abstraction as the mode of alpha-C-H bond cleavage. These results are discussed in terms of several mechanisms proposed for MAO catalysis and with previous structure-activity studies published with bovine liver MAO B [Walker, M. C., and Edmondson, D. E. (1994) Biochemistry 33, 7088-7098].

Anaerobiosis↗
PubMed1999

Influence of flavin analogue structure on the catalytic activities and flavinylation reactions of recombinant human liver monoamine oxidases A and B.

Two riboflavin-deficient (rib5) Saccharomyces cerevisiae expression systems have been developed to investigate the influence of riboflavin structural alterations on the covalent flavinylation reaction and activity of recombinant human liver monoamine oxidases A and B (MAO A and B). Nineteen different riboflavin analogues were tested with MAO A and nine with MAO B. MAO expression and flavinylation were determined immunochemically with antisera to MAO and an anti-flavin antisera. Expression levels of both MAO A and B are invariant with the presence or absence of riboflavin or riboflavin analogues in the growth medium. Flavin analogues with a variety of seven and eight substitutions are found to be covalently incorporated and to confer catalytic activity. The selectivities of MAO A and MAO B for flavin analogue incorporation are found to be similar, although 8alpha-methylation of the flavin resulted in a higher level of catalytic activity for MAO B than for MAO A. N(3)-Methylriboflavin and 8-nor-8-aminoriboflavin are not covalently bound as they are not converted to their respective FAD forms by yeast. 5-Carba-5-deazaflavin and 7,8-nor-7-chlororiboflavin are not covalently incorporated into MAO A and do not support catalytic activity. A flavin peptide was isolated from MAO A containing 7-nor-7-bromo-FAD and was demonstrated to be covalently attached to Cys-406 by an 8alpha-S-thioether linkage by sequence analysis and by matrix-assisted laser desorption ionization time of flight mass spectroscopy. MAO A partially purified from yeast grown on 8-nor-8-chlororiboflavin exhibited an absorption spectrum indicating the covalent flavin is an 8-nor-8-S-thioflavin, suggesting a nucleophilic displacement mechanism that supports the quinone-methide mechanism previously suggested as a general mechanism for covalent flavin attachment.

Base Sequence↗
PubMed1999

The ferroxidase reaction of ferritin reveals a diferric mu-1,2 bridging peroxide intermediate in common with other O2-activating non-heme diiron proteins.

Ferritins are ubiquitous proteins that concentrate, store, and detoxify intracellular iron through oxidation of Fe2+ (ferroxidation), followed by translocation and hydrolysis to form a large inorganic mineral core. A series of mutagenesis, kinetics, and spectroscopic studies of ferritin led to the proposal that the oxidation/translocation path involves a diiron protein site. Recent stopped-flow absorption and rapid freeze-quench Mössbauer studies have identified a single peroxodiferric species as the initial transient intermediate formed in recombinant frog M ferritin during rapid ferroxidation [Pereira, S. A., Small, W., Krebs, C., Tavares, P., Edmondson, D. E., Theil, E. C., and Huynh, B. H. (1998) Biochemistry 37, 9871-9876]. To further characterize this transient intermediate and to establish unambiguously the peroxodiferric assignment, rapid freeze-quenching was used to trap the initial intermediate for resonance Raman investigation. Discrete vibrational modes are observed for this intermediate, indicating a single chromophore in a homogeneous state, in agreement with the Mössbauer conclusions. The frequency at 851 cm-1 is assigned as nu(O-O) of the bound peroxide, and the pair of frequencies at 485 and 499 cm-1 is attributed, respectively, to nus and nuas of Fe-O2-Fe. Identification of the chromophore as a micro-1,2 bridged diferric peroxide is provided by the isotope sensitivity of these Raman bands. Similar peroxodiferric intermediates have been detected in a mutant of the R2 subunit of ribonucleotide reductase from Escherichia coli and chemically reduced Delta9 stearoyl-acyl carrier protein desaturase (Delta9D), but in contrast, the ferritin intermediate is trapped from the true reaction pathway of the native protein. Differences in the Raman signatures of these peroxide species are assigned to variations in Fe-O-O-Fe angles and may relate to whether the iron is retained in the catalytic center or released as an oxidized product.

Animals↗
PubMed1998

Direct spectroscopic and kinetic evidence for the involvement of a peroxodiferric intermediate during the ferroxidase reaction in fast ferritin mineralization.

Rapid freeze-quench (RFQ) Mössbauer and stopped-flow absorption spectroscopy were used to monitor the ferritin ferroxidase reaction using recombinant (apo) frog M ferritin; the initial transient ferric species could be trapped by the RFQ method using low iron loading (36 Fe2+/ferritin molecule). Biphasic kinetics of ferroxidation were observed and measured directly by the Mössbauer method; a majority (85%) of the ferrous ions was oxidized at a fast rate of approximately 80 s-1 and the remainder at a much slower rate of approximately 1.7 s-1. In parallel with the fast phase oxidation of the Fe2+ ions, a single transient iron species is formed which exhibits magnetic properties (diamagnetic ground state) and Mössbauer parameters (DeltaEQ = 1.08 +/- 0.03 mm/s and delta = 0.62 +/- 0.02 mm/s) indicative of an antiferromagnetically coupled peroxodiferric complex. The formation and decay rates of this transient diiron species measured by the RFQ Mössbauer method match those of a transient blue species (lambdamax = 650 nm) determined by the stopped-flow absorbance measurement. Thus, the transient colored species is assigned to the same peroxodiferric intermediate. Similar transient colored species have been detected by other investigators in several other fast ferritins (H and M subunit types), such as the human H ferritin and the Escherichia coli ferritin, suggesting a similar mechanism for the ferritin ferroxidase step in all fast ferritins. Peroxodiferric complexes are also formed as early intermediates in the reaction of O2 with the catalytic diiron centers in the hydroxylase component of soluble methane monooxygenase (MMOH) and in the D84E mutant of the R2 subunit of E. coli ribonucleotide reductase. The proposal that a single protein site, with a structure homologous to the diiron centers in MMOH and R2, is involved in the ferritin ferroxidation step is confirmed by the observed kinetics, spectroscopic properties, and purity of the initial peroxodiferric species formed in the frog M ferritin.

Animals↗
PubMed1998

Reaction of Escherichia coli cytochrome bo3 with substoichiometric ubiquinol-2: a freeze-quench electron paramagnetic resonance investigation.

The reaction of the quinol oxidase cytochrome bo3 from Escherichia coli with ubiquinol-2 (UQ2H2) was carried out using substoichiometric (0.5 equiv) amounts of substrate. Reactions were monitored through the use of freeze-quench EPR spectroscopy. Under 1 atm of argon, semiquinone was formed at the QB site of the enzyme with a formation rate constant of 140 s-1; the QB semiquinone EPR signal decayed with a rate constant of about 5 s-1. Heme b and CuB were reduced within the 10-ms dead time of the freeze-quench experiment and remained at a constant level of reduction over the 1-s time course of the experiment. Quantitation of the reduction levels of QB and heme b during this reaction yielded a reduction potential of 30-60 mV for heme b. Under a dioxygen atmosphere, the rates of semiquinone formation and its subsequent decay were not altered significantly. However, accurate quantitation of the EPR signals for heme b and heme o3 could not be made, due to interference from dioxygen. In the reaction between the QB-depleted enzyme and UQ2H2 under substoichiometric conditions, there was no observable change in the EPR spectra of the enzyme over the time course of the reaction, suggesting an electron transfer from heme b to the binuclear site in the absence of QB which occurs within the dead time of the freeze-quench apparatus. Analysis of the thermodynamics and kinetics of electron transfers in this enzyme suggests that a Q-cycle mechanism for proton translocation is more likely than a cytochrome c oxidase-type ion-pump mechanism.

Cytochrome b Group↗
PubMed1998

The recombinant alpha subunit of glutamate synthase: spectroscopic and catalytic properties.

As part of our studies of Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein, we have overproduced the two enzyme subunits separately in Escherichia coli. The beta subunit (53.2 kDa) was demonstrated to contain the site of NADPH oxidation of glutamate synthase and the FAD cofactor, which was identified as Flavin 1 of glutamate synthase, the flavin located at the site of NADPH oxidation. We now report the overproduction of the glutamate synthase alpha subunit (162 kDa), which is purified to homogeneity in a stable form. This subunit contains FMN as the flavin cofactor which exhibits the properties of Flavin 2 of glutamate synthase: reactivity with sulfite to yield a flavin-N(5)-sulfite addition product (Kd = 2.6 +/- 0.22 mM), lack of reactivity with NADPH, reduction by L-glutamate, and reoxidation by 2-oxoglutarate and glutamine. Thus, FMN is the flavin located at the site of reduction of the iminoglutarate formed on the addition of glutamine amide group to the C(2) carbon of 2-oxoglutarate. The glutamate synthase alpha subunit contains the [3Fe-4S] cluster of glutamate synthase, as shown by low-temperature EPR spectroscopy experiments. The glutamate synthase alpha subunit catalyzes the synthesis of glutamate from L-glutamine and 2-oxoglutarate, provided that a reducing system (dithionite and methyl viologen) is present. The FMN moiety but not the [3Fe-4S] cluster of the subunit appears to participate in this reaction. Furthermore, the isolated alpha subunit of glutamate synthase exhibits a glutaminase activity, which is absent in the glutamate synthase holoenzyme. These findings support a model for glutamate synthase according to which the enzymes prepared from various sources share a common glutamate synthase function (the alpha subunit of the bacterial enzyme, or its homologous polypeptide forming the ferredoxin-dependent plant enzyme) but differ for the chosen electron donor. The pyridine nucleotide-dependent forms of the enzyme have recruited a FAD-dependent oxidoreductase (the bacterial beta subunit) to mediate electron transfer from the NAD(P)H substrate to the glutamate synthase polypeptide. However, it appears that the presence of the enzyme beta subunit and/or of the additional iron-sulfur clusters (Centers II and III) of the bacterial glutamate synthase is required for communication between Center I (the [3Fe-4S] center) and the FMN moiety within the alpha subunit, and for ensuring coupling of glutamine hydrolysis to the transfer of the released ammonia molecule to 2-oxoglutarate in the holoenzyme.

Azospirillum brasilense↗