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Pyranopterin dithiolene distortions relevant to electron transfer in xanthine oxidase/dehydrogenase.

Dong C, Yang J, Leimkühler S, Kirk ML - Inorg Chem (2014)

Bottom Line: The reducing substrates 4-thiolumazine and 2,4-dithiolumazine have been used to form Mo(IV)-product complexes with xanthine oxidase (XO) and xanthine dehydrogenase.The resonance Raman spectra reveal in-plane bending modes of the bound product and low-frequency molybdenum dithiolene and pyranopterin dithiolene vibrational modes.This work provides keen insight into the role of the pyranopterin dithiolene in electron-transfer reactivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, The University of New Mexico , MSC03 2060, 1 University of New Mexico, Albuquerque, New Mexico 87131-0001, United States.

ABSTRACT
The reducing substrates 4-thiolumazine and 2,4-dithiolumazine have been used to form Mo(IV)-product complexes with xanthine oxidase (XO) and xanthine dehydrogenase. These Mo(IV)-product complexes display an intense metal-to-ligand charge-transfer (MLCT) band in the near-infrared region of the spectrum. Optical pumping into this MLCT band yields resonance Raman spectra of the Mo site that are devoid of contributions from the highly absorbing FAD and 2Fe2S clusters in the protein. The resonance Raman spectra reveal in-plane bending modes of the bound product and low-frequency molybdenum dithiolene and pyranopterin dithiolene vibrational modes. This work provides keen insight into the role of the pyranopterin dithiolene in electron-transfer reactivity.

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Left:oxidized and reduced XO/XDH. Right: Reduced “tetrahydro”structure proposed for the pyranopterin dithiolene ligand in the XOfamily of enzymes. The metalated form of pyranopterin dithiolene isoften referred to as the molybdenum cofactor, or Moco.
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fig1: Left:oxidized and reduced XO/XDH. Right: Reduced “tetrahydro”structure proposed for the pyranopterin dithiolene ligand in the XOfamily of enzymes. The metalated form of pyranopterin dithiolene isoften referred to as the molybdenum cofactor, or Moco.

Mentions: Mammalian xanthine oxidoreductase(XOR) and R. capsulatus xanthine dehydrogenase(XDH) are molybdenum hydroxylases with broad substrate specificities.1,2 These enzymes possess a high degree of sequence homology and virtuallyidentical coordination geometries.4,5 Unlike monooxygenases,the oxygen atom incorporated into substrate C–H bonds derivesfrom metal-activated water, and the enzymes generate rather than consumereducing equivalents.2 These reducing equivalentsare transferred sequentially from the reduced MoIV centervia an apparent electron-transfer (ET) chain consisting of the pyranopterinditholene (Figure 1), two 2Fe2S clusters, andFAD.5,9 The ultimate electron acceptor for the oxidaseform of XOR (xanthine oxidase, XO) is O2, and this resultsin the formation of reactive oxygen species that have been implicatedin reperfusion injury following ischemia.2 The ultimate electron acceptor for XDH and the dehydrogenase formof XOR is NAD.4 Integral to the ET regeneration of the catalytically competent MoVI site is the pyranopterin ditholene chelate,9−11 which has been hypothesized to facilitate ET and modulate the molybdenumreduction potential.12,13 The pyranopterin ditholene isone of the most electronically complex ligands in biology,1,9−11,14 containing a redoxnoninnocent dithiolene,14,15 a pyran ring that canexist in both ring-opened16,17 and ring-closed forms,and a redox-active pterin ring system. The Mo ion is not covalentlylinked to the protein but is anchored via the pyranopterin dithiolenethrough an extensive hydrogen-bonding network with the protein. Recently,we showed that pyranopterin ditholene distortions can be correlatedwith enzyme function.11 As a result ofthis analysis, XO family enzymes are proposed to possess a tetrahydropyranopterinditholene (Figure 1) that is intimately involvedin the transfer of redox equivalents from Mo to the proximal 2Fe2Scenter. In spite of the intense interest in metallodithiolenes10 and, more specifically, in the complexity ofthe pyranopterin ditholene,9,10 there is a dearth ofspectroscopic studies that have been directed toward understandinghow the pyranopterin ditholene facilitates ET in XO/XDH. AlthoughXO has been studied by resonance Raman (rR) spectroscopy,18,19 modes attributed to the pyranopterin ditholene have not been assigned.In order to address this issue, we have synthesized new XO/XDH reducingsubstrates that, when oxidized, bind tightly to the MoIV form of the enzyme. The MoIV–product bonding interactionresults in the appearance of an intense near-infrared (NIR) metal-to-ligand(product) charge-transfer (MLCT) band in the electronic absorptionspectrum.18 Specifically, we generatedMoIV-product charge-transfer complexes for bovine XO and R. capsulatus XDH by the enzyme-catalyzed oxidation of 4-thiolumazineand 2,4-dithiolumazine to 4-thioviolapterin (4-TV) and 2,4-dithioviolapterin(2,4-TV), respectively, in a manner similar to that used for the seminalstudies on violapterin.18,20 Alternatively, enzymaticallygenerated product collected and concentrated by centrifugation/filtration,then incubated with reduced XO/XDH, generates the same MoIV-product MLCT complex, as evidenced by electronic absorption spectroscopy.


Pyranopterin dithiolene distortions relevant to electron transfer in xanthine oxidase/dehydrogenase.

Dong C, Yang J, Leimkühler S, Kirk ML - Inorg Chem (2014)

Left:oxidized and reduced XO/XDH. Right: Reduced “tetrahydro”structure proposed for the pyranopterin dithiolene ligand in the XOfamily of enzymes. The metalated form of pyranopterin dithiolene isoften referred to as the molybdenum cofactor, or Moco.
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Related In: Results  -  Collection

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fig1: Left:oxidized and reduced XO/XDH. Right: Reduced “tetrahydro”structure proposed for the pyranopterin dithiolene ligand in the XOfamily of enzymes. The metalated form of pyranopterin dithiolene isoften referred to as the molybdenum cofactor, or Moco.
Mentions: Mammalian xanthine oxidoreductase(XOR) and R. capsulatus xanthine dehydrogenase(XDH) are molybdenum hydroxylases with broad substrate specificities.1,2 These enzymes possess a high degree of sequence homology and virtuallyidentical coordination geometries.4,5 Unlike monooxygenases,the oxygen atom incorporated into substrate C–H bonds derivesfrom metal-activated water, and the enzymes generate rather than consumereducing equivalents.2 These reducing equivalentsare transferred sequentially from the reduced MoIV centervia an apparent electron-transfer (ET) chain consisting of the pyranopterinditholene (Figure 1), two 2Fe2S clusters, andFAD.5,9 The ultimate electron acceptor for the oxidaseform of XOR (xanthine oxidase, XO) is O2, and this resultsin the formation of reactive oxygen species that have been implicatedin reperfusion injury following ischemia.2 The ultimate electron acceptor for XDH and the dehydrogenase formof XOR is NAD.4 Integral to the ET regeneration of the catalytically competent MoVI site is the pyranopterin ditholene chelate,9−11 which has been hypothesized to facilitate ET and modulate the molybdenumreduction potential.12,13 The pyranopterin ditholene isone of the most electronically complex ligands in biology,1,9−11,14 containing a redoxnoninnocent dithiolene,14,15 a pyran ring that canexist in both ring-opened16,17 and ring-closed forms,and a redox-active pterin ring system. The Mo ion is not covalentlylinked to the protein but is anchored via the pyranopterin dithiolenethrough an extensive hydrogen-bonding network with the protein. Recently,we showed that pyranopterin ditholene distortions can be correlatedwith enzyme function.11 As a result ofthis analysis, XO family enzymes are proposed to possess a tetrahydropyranopterinditholene (Figure 1) that is intimately involvedin the transfer of redox equivalents from Mo to the proximal 2Fe2Scenter. In spite of the intense interest in metallodithiolenes10 and, more specifically, in the complexity ofthe pyranopterin ditholene,9,10 there is a dearth ofspectroscopic studies that have been directed toward understandinghow the pyranopterin ditholene facilitates ET in XO/XDH. AlthoughXO has been studied by resonance Raman (rR) spectroscopy,18,19 modes attributed to the pyranopterin ditholene have not been assigned.In order to address this issue, we have synthesized new XO/XDH reducingsubstrates that, when oxidized, bind tightly to the MoIV form of the enzyme. The MoIV–product bonding interactionresults in the appearance of an intense near-infrared (NIR) metal-to-ligand(product) charge-transfer (MLCT) band in the electronic absorptionspectrum.18 Specifically, we generatedMoIV-product charge-transfer complexes for bovine XO and R. capsulatus XDH by the enzyme-catalyzed oxidation of 4-thiolumazineand 2,4-dithiolumazine to 4-thioviolapterin (4-TV) and 2,4-dithioviolapterin(2,4-TV), respectively, in a manner similar to that used for the seminalstudies on violapterin.18,20 Alternatively, enzymaticallygenerated product collected and concentrated by centrifugation/filtration,then incubated with reduced XO/XDH, generates the same MoIV-product MLCT complex, as evidenced by electronic absorption spectroscopy.

Bottom Line: The reducing substrates 4-thiolumazine and 2,4-dithiolumazine have been used to form Mo(IV)-product complexes with xanthine oxidase (XO) and xanthine dehydrogenase.The resonance Raman spectra reveal in-plane bending modes of the bound product and low-frequency molybdenum dithiolene and pyranopterin dithiolene vibrational modes.This work provides keen insight into the role of the pyranopterin dithiolene in electron-transfer reactivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, The University of New Mexico , MSC03 2060, 1 University of New Mexico, Albuquerque, New Mexico 87131-0001, United States.

ABSTRACT
The reducing substrates 4-thiolumazine and 2,4-dithiolumazine have been used to form Mo(IV)-product complexes with xanthine oxidase (XO) and xanthine dehydrogenase. These Mo(IV)-product complexes display an intense metal-to-ligand charge-transfer (MLCT) band in the near-infrared region of the spectrum. Optical pumping into this MLCT band yields resonance Raman spectra of the Mo site that are devoid of contributions from the highly absorbing FAD and 2Fe2S clusters in the protein. The resonance Raman spectra reveal in-plane bending modes of the bound product and low-frequency molybdenum dithiolene and pyranopterin dithiolene vibrational modes. This work provides keen insight into the role of the pyranopterin dithiolene in electron-transfer reactivity.

Show MeSH