Histidinyl Radical Formation in the Self-peroxidation Reaction of Bovine Copper-Zinc Superoxide Dismutase

Hydrogen peroxide can interact with the active site of copper-zinc superoxide dismutase (SOD1) to generate a powerful oxidant. This oxidant can either damage amino acid residues at the active site, inactivating the enzyme (the self-oxidative pathway), or oxidize substrates exogenous to the active site, preventing inactivation (the external oxidative pathway). It is well established that the presence of bicarbonate anion dramatically enhances the rate of oxidation of exogenous substrates. Here, we show that bicarbonate also substantially enhances the rate of self-inactivation of human wild type SOD1. Together, these observations suggest that the strong oxidant formed by hydrogen peroxide and SOD1 in the presence of bicarbonate arises from a pathway mechanistically distinct from that producing the oxidant in its absence. Selfinactivation rates are further enhanced in a mutant SOD1 protein (L38V) linked to the fatal neurodegenerative disorder, familial amyotrophic lateral sclerosis. The 1.4 Å resolution crystal structure of pathogenic SOD1 mutant D125H reveals the mode of oxyanion binding in the active site channel and implies that phosphate anion attenuates the bicarbonate effect by competing for binding to this site. The orientation of the enzyme-associated oxyanion suggests that both the self-oxidative and external oxidative pathways can proceed through an enzyme-associated peroxycarbonate intermediate.

show abstract

“…3B) (41). Histidine copper ligands modified in this way result in copper cofactor loss and enzyme inactivation.…”

Section: Sod-cu(i)(hcomentioning

confidence: 99%

An Alternative Mechanism of Bicarbonate-mediated Peroxidation by Copper-Zinc Superoxide Dismutase

Elam

Malek

Rodríguez

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…In this system, the histidinyl radical reacts with molecular oxygen to form an intermediate peroxyl radical that rapidly decays to form 2-oxohistidine. This oxidized amino acid is implicated in the inactivation of Cu,Zn- superoxide dismutase by its own reaction product, H 2 O 2 (32,33).…”

Section: Mechanism-based Inactivation Of Cox-1 By Resveratrol-mentioning

confidence: 99%

Resveratrol is a Peroxidase-mediated Inactivator of COX-1 but Not COX-2

Szewczuk

Forti

Stivala

et al. 2004

Journal of Biological Chemistry

213

View full text Add to dashboard Cite

Resveratrol (3,4 ,5-trihydroxy-trans-stilbene) is a phytoalexin found in grapes that has anti-inflammatory, cardiovascular protective, and cancer chemopreventive properties. It has been shown to target prostaglandin H 2 synthase (COX)-1 and COX-2, which catalyze the first committed step in the synthesis of prostaglandins via sequential cyclooxygenase and peroxidase reactions. Resveratrol discriminates between both COX isoforms. It is a potent inhibitor of both catalytic activities of COX-1, the desired drug target for the prevention of cardiovascular disease, but only a weak inhibitor of the peroxidase activity of COX-2, the isoform target for nonsteroidal anti-inflammatory drugs. We have investigated the unique inhibitory properties of resveratrol. We find that it is a potent peroxidase-mediated mechanism-based inactivator of COX-1 only (k inact ‫؍‬ 0.069 ؎ 0.004 s ؊1 , K i(inact) ‫؍‬ 1.52 ؎ 0.15 M), with a calculated partition ratio of 22. Inactivation of COX-1 was time-and concentration-dependent, it had an absolute requirement for a peroxide substrate, and it was accompanied by a concomitant oxidation of resveratrol. Resveratrolinactivated COX-1 was devoid of both the cyclooxygenase and peroxidase activities, neither of which could be restored upon gel-filtration chromatography. Inactivation of COX-1 by [ 3 H]resveratrol was not accompanied by stable covalent modification as evident by both SDS-PAGE and reverse phase-high performance liquid chromatography analysis. Structure activity relationships on methoxy-resveratrol analogs showed that the m-hydroquinone moiety was essential for irreversible inactivation of COX-1. We propose that resveratrol inactivates COX-1 by a "hit-and-run" mechanism, and offers a basis for the design of selective COX-1 inactivators that work through a mechanism-based event at the peroxidase active site.

show abstract

“…However, using strongly oxidizing OH ⅐ radicals, generated in a Fenton reaction, transient histidine OHadduct cation (74) and neutral radicals (75) have been generated and investigated in a liquid aqueous solution of histidine. Recently, histidine radicals were also observed in a reaction of superoxide dismutase with hydroperoxide (23). In R2 mutant Y122H, either the oxidation power of the intermediate X (Fe III Fe IV ) is not sufficient for generating a histidine radical, or the hydroxylation of Phe 208 , and subsequent formation of a coordinated phenoxyl radical as a rather stable product, is energetically more favorable.…”

Section: Discussionmentioning

confidence: 99%

“…So far there is no evidence for the involvement of histidine radicals in the catalytic reaction of a protein. However, transient histidine radicals have been generated in Cu/Zn superoxide dismutase during a reaction with hydroperoxide and were indirectly detected by EPR using the spin trap technique (23).…”

mentioning

confidence: 99%

A New Tyrosyl Radical on Phe208 as Ligand to the Diiron Center in Escherichia coli Ribonucleotide Reductase, Mutant R2-Y122H

Kolberg

Logan

Bleifuß

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr 122 ), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, Fe is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe 208 is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.Over the past decades a number of structurally related proteins that contain oxygen-bridged dinuclear iron centers have been discovered and characterized (1, 2). Among these are the hydroxylase protein component of methane monooxygenase (MMO), 1 called MMOH (3-5), and the R2 protein component of class I ribonucleotide reductase (RNR) (6 -8). These proteins reveal a strikingly similar coordination arrangement of their diiron centers, but fulfill entirely different biological functions; in MMOH, the diiron center is located directly at the substrate binding catalytic site and is directly responsible for the hydroxylation of methane or other substrates, whereas in R2, the diiron center is more than 35 Å away from the substrate binding active site located in the second protein subunit R1 (9 -11). The catalytic role of the diiron center in R2 is to generate and stabilize a tyrosyl radical (at position Tyr 122 in Escherichia coli R2), and this tyrosyl radical is connected to the active site in R1 via a chain of H-bonded amino acid residues. It has been proposed that upon substrate binding in R1 Tyr 122 transfers its radical character via this chain to a cysteine (Cys 439 in E. coli R1), which in turn starts substrate turnover from ribonucleotide to deoxyribonucleotide (9 -12).The R2 protein is a homodimer which in its active form contains two -oxo bridged diferric iron centers and a substoichiometric amount of a tyrosyl radical. The active form can be generated in vitro by the so-called reconstitution reaction by adding a 6-fold molar excess of Fe 2ϩ and molecular oxygen to the iron-free apoR2 protein (13). The tyrosyl radical is stabilized by a surrounding cluster of hydrophobic side ch...

show abstract

Histidinyl Radical Formation in the Self-peroxidation Reaction of Bovine Copper-Zinc Superoxide Dismutase

Cited by 38 publications

References 41 publications

An Alternative Mechanism of Bicarbonate-mediated Peroxidation by Copper-Zinc Superoxide Dismutase

An Alternative Mechanism of Bicarbonate-mediated Peroxidation by Copper-Zinc Superoxide Dismutase

Resveratrol is a Peroxidase-mediated Inactivator of COX-1 but Not COX-2

A New Tyrosyl Radical on Phe208 as Ligand to the Diiron Center in Escherichia coli Ribonucleotide Reductase, Mutant R2-Y122H

Contact Info

Product

Resources

About