Flavin and 5‐deazaflavin: A chemical evaluation of ‘modified’ flavoproteins with respect to the mechanisms of redox biocatalysis

Hemmerich, Peter; Massey, Vincent

doi:10.1016/0014-5793(77)81047-8

Cited by 137 publications

(70 citation statements)

References 68 publications

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“…The lack of activity with 1,5-dihydro-5-deaza-FMN described here strongly indicates the involvement of flavin in catalysis by the E. coli enzyme (33). A similar result has previously been SCHEME 2. reported for the bifunctional N. crassa enzyme (15), which possesses an additional NADPH:FMN oxidoreductase activity (3).…”

Section: Discussionsupporting

confidence: 88%

Studies with Substrate and Cofactor Analogues Provide Evidence for a Radical Mechanism in the Chorismate Synthase Reaction

Osborne¹,

Thorneley²,

Abell³

et al. 2000

Journal of Biological Chemistry

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Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The strict requirement for a reduced FMN cofactor and a trans-1,4-elimination are unusual. (6R)-6-Fluoro-EPSP was shown to be converted to chorismate stoichiometrically with enzyme-active sites in the presence of dithionite. This conversion was associated with the oxidation of FMN to give a stable flavin semiquinone. The IC 50 of the fluorinated substrate analogue was 0.5 and 250 M with the Escherichia coli enzyme, depending on whether it was preincubated with the enzyme or not. The lack of dissociation of the flavin semiquinone and chorismate from the enzyme appears to be the basis of the essentially irreversible inhibition by this analogue. A dithionite-dependent transient formation of flavin semiquinone during turnover of (6S)-6-fluoro-EPSP has been observed. These reactions are best rationalized by radical chemistry that is strongly supportive of a radical mechanism occurring during normal turnover. The lack of activity with 5-deaza-FMN provides additional evidence for the role of flavin in catalysis by the E. coli enzyme.Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) 1 to chorismate (Scheme 1) (1). This enzyme is the seventh enzyme of the shikimate pathway and is present in bacteria, fungi, and plants but not in mammals. It is therefore a potential target for antimicrobial agents and herbicides. The strict requirement for a reduced FMN cofactor (2-4) and the trans-1,4-elimination of the 3-phosphate and the 6R-H (5-7) are both unusual aspects of the chorismate synthase reaction. A number of non-concerted mechanisms have been proposed to account for these properties (1,8,9).Evidence for a non-concerted mechanism includes a secondary tritium kinetic isotope effect at C(3) (10), the slow conversion of (6S)-6-fluoro-EPSP to 6-fluoro-chorismate (11), transient kinetics studies (12), and more recently, a secondary ␤ deuterium kinetic isotope effect at C(4) (13). These studies collectively make the radical (Scheme 1A) and E1 (Scheme 1B) mechanisms involving the initial cleavage of the C(3)-O bond the most likely, and alternative mechanisms where the C(6)-H bond breaks first the least likely (8,14). Evidence for the radical mechanism (Scheme 1A), which provides a role for the reduced flavin in catalysis, comes from the lack of activity of the bifunctional Neurospora crassa enzyme with 5-deaza-FMN (15) and the formation of a flavin semiquinone with the monofunctional Escherichia coli enzyme and the inhibitor (6R)-6-fluoro-EPSP (16).This paper describes new studies with the E. coli enzyme that are strongly supportive of the proposed radical mechanism (Scheme 1A). The product of the reaction with the inhibitor (6R)-6-fluoro-EPSP has been identified, allowing the formation of the stable flavin semiquinone radical in the presence of dithionite to be rationalized. The formation of a flavin semiquinone radical during turnover of (6S)-6-fluoro-EPSP has been observed in the prese...

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Section: Discussionsupporting

confidence: 88%

Studies with Substrate and Cofactor Analogues Provide Evidence for a Radical Mechanism in the Chorismate Synthase Reaction

Osborne¹,

Thorneley²,

Abell³

et al. 2000

Journal of Biological Chemistry

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“…A more detailed classification has been proposed in a recent paper by Hemmerich et al [33] who distinguish four classes of flavoproteins according to the nature of both input and output reactions. Of relevance to the present discussion is the concept of dehydrogenasesoxidases and dehydrogenases-electron transferases, with identical type of input activity, but different output reactions.…”

Section: Discussionmentioning

confidence: 99%

Sulfite Binding to a Flavodehydrogenase, Cytochrome b₂ from Baker's Yeast

Lederer

1978

European Journal of Biochemistry

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Baker's yeast L-lactate dehydrogenase (flavocytochrome b2) is a typical flavodehydrogenase, in that it accepts two electrons from the substrate but has a monoelectronic acceptor. Yet it forms a red semiquinone [Capeillere Blandin et al. Eur. J. Bioclwm. 54, 549-566 (1975)j and it is shown in this paper that it forms a reversible covalent complex with sulfite (Kd = 1.4 pM). This complex can be observed by difference spectroscopy and provides a convenient tool for visualizing the flavin chromophore, usually hidden behind the intense heme absorbance. A number of anions (D-lactate, oxalate and pyruvate) are inhibitors of the enzymatic reaction and induce spectral perturbations of the flavin spectrum. It is concluded that probably two positive charges exist at the active site: one which stabilizes the red semiquinone and one which attracts organic anions and sulfite. It is also concluded that the correlation between reactivity with sulfite and reactivity with oxygen among flavoproteins may not be as general as previously proposed [Massey et al. J. Biol. Chem. 244,[3999][4000][4001][4002][4003][4004][4005][4006] (1 969)].Free reduced flavin is rapidly reoxidized by oxygen, whereas enzyme-bound flavin either keeps or loses this property, according to the function of the protein. This phenomenon provides the basis for the now classical distinction, among flavoproteins, between oxidases and dehydrogenases. It is clear that the protein moiety plays a fundamental role in modulating the reactivity of the bound flavin, but this role is at present poorly understood, in spite of numerous efforts.A few years ago, Massey et al. [l] proposed an interesting correlation between the reactivity of flavoproteins toward oxygen and toward sulfite. It was shown that a number of flavoproteins were bleached by sulfite, which reversibly formed a covalent bond with N-5 of the flavin nucleus [2]; those proteins that reacted were oxidases and formed anionic (red) semiquinones; those that did not react were dehydrogenases and formed neutral (blue) semiquinones. A tentative explanation was presented, based on these and other observations: a positive charge at the active site of oxidases would facilitate sulfite binding and stabilize the anionic semiquinone ;

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“…The first report dealing with the role of a specific flavin function was that by Edmondson et al, which addressed the role of N(5) and involved the use of 5-deaza-FMN and a flavodoxin [16]. It was followed by a long series of papers dealing with the same 5-deazaflavin model and its use for the study of biochemical mechanisms (see [1] and [17] for reviews on 5-deazaflavins). Studies on the interactions of flavin with the protein active centre by using spectral probes were initiated by the use of the naturally occurring 6-OH and 8-OH-flavin chromophores [18,19].…”

Section: Modes Of Interaction Of Flavin With Proteinmentioning

confidence: 99%

“…Thermodynamically the reaction of reduced 5-deazaflavin with oxygen is even more favourable than that with normal reduced flavin (EO =-300 mV versus -210 mV). The [17].…”

Section: Mechanism Probesmentioning

confidence: 99%

New flavins for old: artificial flavins as active site probes of flavoproteins

Ghisla¹,

Massey²

1986

Biochemical Journal

195

143

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Flavin and 5‐deazaflavin: A chemical evaluation of ‘modified’ flavoproteins with respect to the mechanisms of redox biocatalysis

Cited by 137 publications

References 68 publications

Studies with Substrate and Cofactor Analogues Provide Evidence for a Radical Mechanism in the Chorismate Synthase Reaction

Studies with Substrate and Cofactor Analogues Provide Evidence for a Radical Mechanism in the Chorismate Synthase Reaction

Sulfite Binding to a Flavodehydrogenase, Cytochrome b₂ from Baker's Yeast

New flavins for old: artificial flavins as active site probes of flavoproteins

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Flavin and 5‐deazaflavin: A chemical evaluation of ‘modified’ flavoproteins with respect to the mechanisms of redox biocatalysis

Cited by 137 publications

References 68 publications

Studies with Substrate and Cofactor Analogues Provide Evidence for a Radical Mechanism in the Chorismate Synthase Reaction

Studies with Substrate and Cofactor Analogues Provide Evidence for a Radical Mechanism in the Chorismate Synthase Reaction

Sulfite Binding to a Flavodehydrogenase, Cytochrome b2 from Baker's Yeast

New flavins for old: artificial flavins as active site probes of flavoproteins

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Product

Resources

About

Sulfite Binding to a Flavodehydrogenase, Cytochrome b₂ from Baker's Yeast