General magnetic dipolar interaction of spin-spin coupled molecular dimers.  Application to an EPR spectrum of xanthine oxidase

Coffman, Robert E.; Buettner, Garry R.

doi:10.1021/j100481a018

Cited by 55 publications

(20 citation statements)

References 3 publications

(3 reference statements)

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“…The crystal structure shows iron a at 7.8 Å from the 7␣-methyl carbon of the flavin ring and at 12.4 Å from the nearest iron atom of the Fe͞S I cluster, both distances significantly shorter than those between the Cys 51-liganded iron atom b and the nearest atoms in the FAD and Fe͞S I cofactors (8.1 Å and 14.1 Å, respectively). The distance between the Mo-ion and the nearest iron atom in the Fe͞S I cluster is 14.7 Å, confirming the estimate of 14 Å based on the measurement of dipolar interaction between the two paramagnetic centers (33). The geometrical arrangements and redox potentials of these centers indicate that electrons are transferred from Mo to the two Fe͞S centers in a thermodynamically favorable process.…”

Section: Resultssupporting

confidence: 80%

Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion

Enroth

Eger²,

Okamoto³

et al. 2000

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

643

601

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Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. Here, we present the crystal structure of the dimeric (Mr, 290,000) bovine milk XDH at 2.1-Å resolution and XO at 2.5-Å resolution and describe the major changes that occur on the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20-kDa domain containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423OLys 433). This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme. Milk xanthine oxidase is an archetypal enzyme, which was originally described as aldehyde oxidase in 1902 (1) and has since served as a benchmark for the whole class of complex metalloflavoproteins (2). Xanthine oxidoreductase enzymes have been isolated from a wide range of organisms, from bacteria to man, and catalyze the hydroxylation of a wide variety of purine, pyrimidine, pterin, and aldehyde substrates. All of these proteins have similar molecular weights and composition of redox centers (3, 4). The mammalian enzymes, which catalyze the hydroxylation of hypoxanthine and xanthine, the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) and exist mostly as such in the cell but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. XDH shows a preference for NAD ϩ reduction at the flavin adenine dinucleotide (FAD) reaction site, whereas XO fails to react with NAD ϩ and exclusively uses dioxygen as its substrate, leading to the formation of superoxide anion and hydrogen peroxide (3). The enzyme is a target of drugs against gout and hyperuricemia (5), and the conversion of XDH to XO is of major interest as it has been implicated in diseases characterized by oxygen-radical-induced tissue damage, such as postischemic reperfusion injury (6). Recent work suggests that XO also might be associated with blood pressure regulation (7).The active form of the enzyme is that of a homodimer of molecular mass 290 kDa, with each of the monomers acting independently in catalysis. Each subunit contains one molybdopterin cofactor, two spectroscopically distinct [2Fe-2S] centers, and one FAD cofactor. The oxidation of xanthine takes place at the molybdopterin center (Mo-pt) and the electrons thus introduced are rapidly distributed to the other c...

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Section: Resultssupporting

confidence: 80%

Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion

Enroth

Eger²,

Okamoto³

et al. 2000

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

643

601

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“…This dependence argues for spin-spin interactions between these two species with exchange and dipolar contributions (10,15,16). Such magnetic interactions can be observed in cw EPR spectra between paramagnetic centers localized less than 15 Å apart, suggesting that these centers in Hase I are adjacent to each other (17) (Fig. 1).…”

Section: Resultsmentioning

confidence: 87%

“…3 B and C) is accompanied by the formation of a "splitting" of the Ni-B signals (Fig. 4A), indicative of a magnetic interaction between these two centers (15,17). When the sample is annealed to 80 K the uncoupled EPR spectrum of the Ni-B state is recovered, due to the faster relaxation of [FeS] clusters at such temperatures (Fig.…”

Section: Assignment Of the [Fes]mentioning

confidence: 94%

“…1, Fig. S1) and is too large for these two species to exhibit magnetically coupled EPR spectra (17). Therefore, the complex signals are attributed to an interaction pattern of the two reduced proximal and distal clusters (S=1∕2) mediated by the high spin ½3Fe4S 0 cluster (S ¼ 2).…”

Section: Assignment Of the [Fes]mentioning

confidence: 99%

“…These spectral features show that the paramagnetic HP species must be positioned sufficiently close to both the [NiFe] and the [3Fe4S] cluster to allow magnetic interactions. The HP center is thus identified as the proximal iron-sulfur cluster lying in between these centers (8,15,17), implying that it has become oxidized to the 3þ state.…”

Section: Assignment Of the [Fes]mentioning

confidence: 99%

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Characterization of a unique [FeS] cluster in the electron transfer chain of the oxygen tolerant [NiFe] hydrogenase from Aquifex aeolicus

Pandelia

Nitschke

Infossi

et al. 2011

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

153

198

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Iron-sulfur clusters are versatile electron transfer cofactors, ubiquitous in metalloenzymes such as hydrogenases. In the oxygentolerant Hydrogenase I from Aquifex aeolicus such electron "wires" form a relay to a diheme cytb, an integral part of a respiration pathway for the reduction of O 2 to water. Amino acid sequence comparison with oxygen-sensitive hydrogenases showed conserved binding motifs for three iron-sulfur clusters, the nature and properties of which were unknown so far. Electron paramagnetic resonance spectra exhibited complex signals that disclose interesting features and spin-coupling patterns; by redox titrations three iron-sulfur clusters were identified in their usual redox states, a ½3Fe4S and two ½4Fe4S , but also a unique high-potential (HP) state was found. On the basis of 57 Fe Mössbauer spectroscopy we attribute this HP form to a superoxidized state of the [4Fe4S] center proximal to the [NiFe] site. The unique environment of this cluster, characterized by a surplus cysteine coordination, is able to tune the redox potentials and make it compliant with the ½4Fe4S 3þ state. It is actually the first example of a biological [4Fe4S] center that physiologically switches between 3þ, 2þ, and 1þ oxidation states within a very small potential range. We suggest that the (1 þ ∕2þ) redox couple serves the classical electron transfer reaction, whereas the superoxidation step is associated with a redox switch against oxidative stress.electrochemistry | EPR | iron-sulfur centers | O2-sensitivity H ydrogenases are metalloproteins occurring in the metabolic pathway of a wide variety of microbial organisms and catalyze the reversible oxidation of dihydrogen: H 2 ⇌ 2H þ þ 2e − (1). The growing interest in alternative sources of energy has focused scientific research on understanding and engineering these enzymes for future applications (2). One of the major limitations of hydrogenases, however, is their sensitivity towards oxygen. Recently, the discovery of hydrogenases that retain catalytic activity in oxygenic environments has potentially opened new applications as "green" vanguard catalysts, in particular as electrocatalysts on electrodes for biofuel cells (3,4).Aquifex aeolicus is a hyperthermophilic Knallgas bacterium with optimum growth temperature of 85°C (5). This microorganism harbors three distinct [NiFe] hydrogenases, among which Hase I is located in the aerobic respiration pathway and attached to the membrane via a diheme cytb (6). Hase I consists of two subunits; the large subunit contains the hetero-bimetallic nickel-iron site and the small subunit the electron transfer cofactors, namely iron-sulfur clusters (6). Based on spectroelectrochemical studies, this enzyme exhibits enhanced thermostability and tolerance for inhibitors (e.g., O 2 and CO) (4, 7).Although the structures of O 2 -sensitive hydrogenases are well characterized (8, 9), such information is still lacking for O 2 -tolerant enzymes. The molecular mechanism and structural determinants for this increased oxygen tolerance remain to ...

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Xanthine Oxidoreductase

Nishino

Pai

2004

Handbook of Metalloproteins

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A single gene product has been identified as responsible for the two catalytic activities, xanthine dehydrogenase, XDH (EC 1.1.1.204), and xanthine oxidase, XO (EC 1.2.3.2). Synthesized as XDH (ultimate electron acceptor NAD + ), the protein can be converted to XO (ultimate electron acceptor oxygen) either reversibly by oxidation of cysteine residues or irreversibly by proteolysis. Xanthine oxidoreductase (XOR) is a ubiquitous enzyme; its physiological role is the oxidation of hypoxanthine to xanthine and further to uric acid, although in vitro the enzyme's specificity is rather broad, accepting a large number of purines, pteridines, and aldehydes as substrates. XOR, in contrast to most other hydroxylases, incorporates water‐derived oxygen into its substrate. The electrons are transferred from xanthine to a Mopterin center and from there via two Fe 2 –S 2 clusters and FAD to NAD + or oxygen, respectively. The enzyme is the target of the antigout drug allopurinol; it is involved in hyperuricemia and xanthinuria and postulated to participate in postischemic reperfusion injury. Crystal structures have been determined for native and mutant XDHs and XOs from man, cow, rat, and Rhodobacter capsulatus . The conformational changes linked to the dehydrogenase/oxidase transition have been identified and interpreted. A structure‐based explanation for the catalytic mechanism is emerging.

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General magnetic dipolar interaction of spin-spin coupled molecular dimers. Application to an EPR spectrum of xanthine oxidase

Cited by 55 publications

References 3 publications

Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion

Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion

Characterization of a unique [FeS] cluster in the electron transfer chain of the oxygen tolerant [NiFe] hydrogenase from Aquifex aeolicus

Xanthine Oxidoreductase

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