Reversible H atom abstraction catalyzed by the radical S-adenosylmethionine enzyme HydG - PubMed (original) (raw)

. 2014 Sep 24;136(38):13086-9.

doi: 10.1021/ja504618y. Epub 2014 Sep 10.

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Reversible H atom abstraction catalyzed by the radical S-adenosylmethionine enzyme HydG

Benjamin R Duffus et al. J Am Chem Soc. 2014.

Abstract

The organometallic H-cluster at the active site of [FeFe]-hydrogenases is synthesized by three accessory proteins, two of which are radical S-adenosylmethionine enzymes (HydE, HydG) and one of which is a GTPase (HydF). In this work we probed the specific role of H atom abstraction in HydG-catalyzed carbon monoxide and cyanide production from tyrosine. The isotope distributions of 5'-deoxyadenosine and p-cresol were evaluated using deuterium-labeled tyrosine substrates in H2O and D2O. The observation of multiply deuterated 5'-deoxyadenosine and deuterated S-adenosylmethionine when the reaction is carried out in D2O provides evidence for a 5'-deoxyadenosyl radical-mediated abstraction of a hydrogen atom from a solvent-exchangeable position as a reversible event.

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Figures

Figure 1

Figure 1

[FeFe]-hydrogenase H-cluster active site from Clostridium pasteurianum (PDB: 3C8Y).

Figure 2

Figure 2

Reaction catalyzed by HydG. Top: Conversion of tyrosine to _p_-cresol, CO, and CN−. Concomitant with this reaction, SAM is converted to methionine and 5′-deoxyadenosine. Bottom: Two [4Fe-4S] clusters are involved. The N-terminal cluster binds and reductively cleaves SAM, and is required for the cleavage of tyrosine to produce _p_-cresol. The C-terminal cluster is important for diatomic ligand production, and the unique iron of this cluster may provide a site for diatomic ligand coordination prior to transfer to HydF. The cysteine motifs coordinating each cluster are shown below the cluster.

Figure 3

Figure 3

ESI-MS of HydG reaction product dAdoH for assays performed in 95% D2O buffer (50 mM tris, pD 8.1). (A) Full reaction. (B) dAdoH reference sample in H2O. Spectra are represented as normalized, extracted ion chromatograms.

Figure 4

Figure 4

ESI-MS of SAM for HydG assays performed in 95% D2O buffer (50 mM tris, pD 8.1). (A) Full reaction. (B) SAM reference sample in H2O. Spectra are represented as normalized, extracted ion chromatograms.

Figure 5

Figure 5

Quantitative _p_-cresol product detection in tris-H2O (■) and 95% tris-D2O (▲). Assays contained 40 μM HydG (9.5 ± 0.2 Fe/protein), 1 mM SAM, 1 mM Tyr, 5 mM dithionite, performed at 37 °C in 50 mM tris, 300 mM KCl, pH/pD 8.1.

Figure 6

Figure 6

Mechanistic proposals for observed HydG H atom abstraction—reabstraction events and dAdo• regeneration. Hydrogen atoms that have undergone exchange with solvent are colored blue.

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References

    1. Vignais P.; Billoud B. Chem. Rev. 2007, 107, 4206. - PubMed
    1. Peters J. W.; Lanzilotta W. N.; Lemon B. J.; Seefeldt L. C. Science 1998, 282, 1853. - PubMed
    1. McGlynn S. E.; Shepard E. M.; Winslow M. A.; Naumov A. V.; Duschene K. S.; Posewitz M. C.; Broderick W. E.; Broderick J. B.; Peters J. W. FEBS Lett. 2008, 582, 2183. - PubMed
    1. Shepard E. M.; McGlynn S. E.; Bueling A. L.; Grady-Smith C. S.; George S. J.; Winslow M. A.; Cramer S. P.; Peters J. W.; Broderick J. B. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 10448–10453. - PMC - PubMed
    1. Posewitz M. C.; King P. W.; Smolinski S. L.; Zhang L.; Seibert M.; Ghirardi M. L. J. Biol. Chem. 2004, 279, 25711. - PubMed

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