Nicotinamide‐Dependent One‐Electron and Two‐Electron (Flavin) Oxidoreduction : Thermodynamics, Kinetics, and Mechanism

Blankenhorn, Gunter

doi:10.1111/j.1432-1033.1976.tb10634.x

Cited by 121 publications

(65 citation statements)

References 62 publications

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“…Comparison of the reactivity of GTN with reduced flavins and reduced deazaflavins does, however, give support to the radical pathway of Scheme 1B. Because of the thermodynamic properties of 5-deazaflavins (26), with redox potentials of ϷϪ650 mV for the oxidized/semiquinone (ox͞sq) couple, ϷϪ300 mV for the ox͞red couple, and Ϸϩ50 mV for the sq͞red couple, 5-deazaflavins generally are considered as incompetent in radical reaction pathways (30,31). On the other hand, they are quite competent for hydride transfer, and in the case of acyl CoA dehydrogenase, the reduced 5-deazaFAD enzyme form was found to be oxidized by crotonyl CoA some 4 times faster than normal enzyme (32).…”

Section: Discussionmentioning

confidence: 99%

Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

Meah

Brown

Chakraborty

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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The reaction of the old yellow enzyme and reduced flavins with organic nitrate esters has been studied. Reduced flavins have been found to react readily with glycerin trinitrate (GTN ) (nitroglycerin) and propylene dinitrate, with rate constants at pH 7.0, 25°C of 145 M ؊1 s ؊1 and 5.8 M ؊1 s ؊1 , respectively. With GTN, the secondary nitrate was removed reductively 6 times faster than the primary nitrate, with liberation of nitrite. With propylene dinitrate, on the other hand, the primary nitrate residue was 3 times more reactive than the secondary residue. In the old yellow enzyme-catalyzed NADPH-dependent reduction of GTN and propylene dinitrate, ping-pong kinetics are displayed, as found for all other substrates of the enzyme. Rapid-reaction studies of mixing reduced enzyme with the nitrate esters show that a reduced enzyme-substrate complex is formed before oxidation of the reduced flavin. The rate constants for these reactions and the apparent Kd values of the enzyme-substrate complexes have been determined and reveal that the rate-limiting step in catalysis is reduction of the enzyme by NADPH. Analysis of the products reveal that with the enzymecatalyzed reactions, reduction of the primary nitrate in both GTN and propylene dinitrate is favored by comparison with the freeflavin reactions. This preferential positional reactivity can be rationalized by modeling of the substrates into the known crystal structure of the enzyme. In contrast to the facile reaction of free reduced flavins with GTN, reduced 5-deazaflavins have been found to react some 4 -5 orders of magnitude slower. This finding implies that the chemical mechanism of the reaction is one involving radical transfers.

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Section: Discussionmentioning

confidence: 99%

Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

Meah

Brown

Chakraborty

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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“…The majority of the cluster can be reduced to [4Fe-4S] 1ϩ , but the reduction requires strongly reducing conditions. FeS clusters requiring photochemical reduction by deazaflavin semiquinones may have potentials for the 2 ϩ /1 ϩ couple as low as Ϫ600 mV (20), far below any cellular reducing power. EPR spectra of [4Fe-4S] 1ϩ clusters may vary with their solvent environment (41), reflecting different delocalization of electrons within the cluster.…”

Section: Discussionmentioning

confidence: 99%

“…The potential of the 2ϩ/1ϩ couple is variable among known [4Fe-4S] clusters. Clusters with very low reduction potentials (ϳ Ϫ600 mV) (20) may be photoreduced by deazariboflavin in the presence of EDTA, a hydrogen atom donor (21)(22)(23). The 2ϩ HiPiPs are readily oxidized by ferricyanide (24).…”

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confidence: 99%

Redox Reactions of the Iron-Sulfur Cluster in a Ribosomal RNA Methyltransferase, RumA

Agarwalla

Stroud

Gaffney

2004

Journal of Biological Chemistry

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RumA, an S-adenosyl-L-methionine-dependent methyltransferase specifically catalyzes the methylation of U1939 of 23 S ribosomal RNA to yield 5-methyluridine (m 5 U) 1 or ribothymidine (1). This enzyme was found to contain a [4Fe-4S] cluster, a prosthetic group usually not associated with methyltransferase enzymes. The x-ray structure of RumA revealed that the cluster is located in the RNA binding domain of the threedomain protein (2). It is held by four Cys-iron bonds provided by Cys 81 , Cys 87 , Cys 90 , and Cys 162 and is buried in a largely hydrophobic region, but one of its sulfur atoms is quite exposed to solvent. The closest distance between the cluster and the sulfur of the catalytic nucleophile, Cys 389 , is ϳ19 Å. RumA is a member of a growing list of nucleic acid modifying enzymes where the function of the iron-sulfur (FeS) cluster remains enigmatic.The reaction mechanism of m 5 U methyltransferases is similar to that of thymidylate synthase, and has been studied mainly employing the tRNA m 5 U54 methyltransferase, TrmA (3-5). It involves Michael addition of the catalytic Cys to carbon 6 (C-6) of pyrimidine, which activates C-5 for a nucleophilic attack on the methyl group of S-adenosyl-L-methionine. Following the methyl transfer, the enzyme is resolved by abstraction of a proton from C-5 and ␤-elimination. TrmA, as well as DNA and RNA 5-methylcytidine methyltransferases, which employ a similar catalytic mechanism (6), do not posses an FeS cluster or any other prosthetic group. Lack of a redox step in the catalytic mechanism, and absence of the cluster in enzymes that employ a similar mechanism preclude a direct role for [4Fe-4S] cluster in the RumA-catalyzed reaction.Two features distinguish the FeS cluster in RumA from the clusters in a different class of S-adenosyl-L-methionine-dependent enzymes that proceed by free radical mechanisms. The RumA cluster is ligated by 4 cysteines and the iron-binding sequence motif is CX 5 CX 2 CX n C. In contrast, the radical Sadenosyl-L-methionine enzymes, of which MiaB (7) and biotin synthase (8) are examples, have a [4Fe-4S] cluster built of a CX 3 CX 2 C sequence that leaves one iron of the cluster free to participate in catalysis. MiaB is a member of a family of enzymes catalyzing addition of sulfur to tRNA bases (9). The FeS sequence motifs in the DNA repair enzymes, endonuclease III (10) and MutY (11), are CX 6 CX 2 CX 5 C, somewhat similar to the RumA sequence. Although the FeS clusters in these enzymes are usually regarded as structural, recently it was shown that the redox potential of MutY is altered by DNA binding and that a change in charge of the [4Fe-4S] 3ϩ/2ϩ cluster might alter its affinity for DNA (12).The redox potentials of [4Fe-4S] clusters vary widely (13-16). Of particular interest in many contemporary studies of FeS proteins is understanding the factors that influence the potentials, and thus functions, of these clusters (17,18). Proteins with four Cys-iron bonds to the [4Fe-4S] cluster fall into two general redox groups, ones with an accessib...

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“…If a covalent adduct was to be formed as shown in Fig. 13 [52]. Importantly, however, in no case has a covalent adduct ever been observed.…”

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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Nicotinamide‐Dependent One‐Electron and Two‐Electron (Flavin) Oxidoreduction : Thermodynamics, Kinetics, and Mechanism

Cited by 121 publications

References 62 publications

Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

Redox Reactions of the Iron-Sulfur Cluster in a Ribosomal RNA Methyltransferase, RumA

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

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