Heterogeneity of the Local Electrostatic Environment of the Tyrosyl Radical in <i>Mycobacterium tuberculosis</i> Ribonucleotide Reductase Observed by High-Field Electron Paramagnetic Resonance

Liu, Aimin; Barra, Anne-Laure; Rubin, Harvey; Lu, Guizhen; Gräslund, Astrid

doi:10.1021/ja990123n

Cited by 28 publications

(22 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…; Table S5) (16)(17)(18)(19)(20)(21). A similar g-tensor anisotropy effect would be expected for a hydroxylated Trp radical because the radical spin density is typically delocalized over the entire indole ring (22)(23)(24)(25)(26)(27).…”

Section: Resultsmentioning

confidence: 53%

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Yukl

Liu

Krzystek

et al. 2013

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

Self Cite

View full text Add to dashboard Cite

Despite the importance of tryptophan (Trp) radicals in biology, very few radicals have been trapped and characterized in a physiologically meaningful context. Here we demonstrate that the diheme enzyme MauG uses Trp radical chemistry to catalyze formation of a Trp-derived tryptophan tryptophylquinone cofactor on its substrate protein, premethylamine dehydrogenase. The unusual sixelectron oxidation that results in tryptophan tryptophylquinone formation occurs in three discrete two-electron catalytic steps. Here the exact order of these oxidation steps in the processive six-electron biosynthetic reaction is determined, and reaction intermediates are structurally characterized. The intermediates observed in crystal structures are also verified in solution using mass spectrometry. Furthermore, an unprecedented Trp-derived diradical species on premethylamine dehydrogenase, which is an intermediate in the first two-electron step, is characterized using high-frequency and -field electron paramagnetic resonance spectroscopy and UV-visible absorbance spectroscopy. This work defines a unique mechanism for radical-mediated catalysis of a protein substrate, and has broad implications in the areas of applied biocatalysis and understanding of oxidative protein modification during oxidative stress.cofactor biosynthesis | tryptophan radical | heme | posttranslational modification | electron transfer P rotein-based radicals, particularly on tyrosine (Tyr) and tryptophan (Trp) residues, have been implicated in a large number of catalytic and electron transfer reactions in biology (1), including the long-range electron transfer reactions required for photosynthesis (2), respiration (3), and DNA synthesis (4) and repair (5). Aberrant formation of protein radicals during oxidative stress is also of special importance. Evidence for the involvement of protein-based and substrate-based radicals in enzyme-catalyzed reactions has increased substantially in recent years, and expanded our knowledge of the scope of chemical reactions accessible to enzymes. The ability of enzymes to catalyze what were previously thought to be unattainable reactions is exemplified by MauG. The biosynthesis of the tryptophan tryptophylquinone (TTQ) cofactor in methylamine dehydrogenase (MADH) requires posttranslational modification of two tryptophan residues. MADH from Paracoccus denitrificans is a 119-kDa α 2 β 2 heterotetramer that contains two active sites and two TTQ cofactors derived from residues βTrp57 and βTrp108 (6). MauG is a c-type diheme enzyme that catalyzes the conversion of a MADH precursor (preMADH) that has one oxygen atom already inserted into the βTrp57 indole ring (βTrp57-OH of preTTQ) (7), to mature TTQ-containing MADH (8) (Fig. 1).Catalysis by MauG proceeds via a bis-Fe(IV) redox state, which may be generated by reaction of di-Fe(II) MauG with O 2 or di-Fe(III) MauG with H 2 O 2 (9). Catalytically active crystals of the MauG-preMADH protein complex show that the site of TTQ formation on β-preMADH is 40.1 Å from the MauG highspin heme iron...

show abstract

Section: Resultsmentioning

confidence: 53%

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Yukl

Liu

Krzystek

et al. 2013

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

Self Cite

View full text Add to dashboard Cite

show abstract

“…Table 1 summarizes g-tensors for tyrosyl radicals from different selected class I RNRs. It was shown theoretically and experimentally that the g x -value is sensitive to the electrostatic environment of the tyrosyl radical in particular to hydrogen bonds between the CϭO group of the tyrosyl radical and the protein environment (6,(52)(53)(54)(55)(56)(57). The g x -value of 2.00916 (5) obtained for the tyrosyl radical Tyr-105⅐ in R2F and R1E⅐R2F of S. typhimurium clearly belongs to the first type of tyrosyl radicals as in E. coli, which is not hydrogenbonded.…”

Section: Frozen Solution W-band Epr Ofmentioning

confidence: 97%

Orientation of the Tyrosyl Radical inSalmonella typhimuriumClass Ib Ribonucleotide Reductase Determined by High Field EPR of R2F Single Crystals

Galander¹,

Uppsten

Uhlin

et al. 2006

J. Biol. Chem.

View full text Add to dashboard Cite

The R2 protein of class I ribonucleotide reductase (RNR) generates and stores a tyrosyl radical, located next to a diferric iron center, which is essential for ribonucleotide reduction and thus DNA synthesis. X-ray structures of class Ia and Ib proteins from various organisms served as bases for detailed mechanistic suggestions. The active site tyrosine in R2F of class Ib RNR of Salmonella typhimurium is located at larger distance to the diiron site, and shows a different side chain orientation, as compared with the tyrosine in R2 of class Ia RNR from Escherichia coli. No structural information has been available for the active tyrosyl radical in R2F. Here we report on high field EPR experiments of single crystals of R2F from S. typhimurium, containing the radical Tyr-105 ⅐ . Full rotational pattern of the spectra were recorded, and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical Tyr-105 ⅐ in the crystal frame. Comparison with the orientation of the reduced tyrosine Tyr-105-OH from the x-ray structure reveals a rotation of the tyrosyl side chain, which reduces the distance between the tyrosyl radical and the nearest iron ligands toward similar values as observed earlier for Tyr-122 ⅐ in E. coli R2. Presence of the substrate binding subunit R1E did not change the EPR spectra of Tyr-105 ⅐ , indicating that binding of R2E alone induces no structural change of the diiron site. The present study demonstrates that structural and functional information about active radical states can be obtained by combining x-ray and high-field-EPR crystallography. Ribonucleotide reductases (RNRs)3 catalyze the reduction of all four ribonucleotides to the corresponding deoxyribonucleotides. This reaction is the rate-limiting step in the DNA synthesis (1-5). In all RNRs investigated so far, a presumed transient thiyl radical in the large protein subunit near the bound substrate (Cys-439, Escherichia coli numbering) is supposed to start the substrate turnover by the abstraction of the 3Ј-H from the ribonucleotide. The substrate turnover proceeds through a series of intermediate states, some of which are radicals. The thiyl radical is recovered, after the product (deoxyribonucleotide) is formed and released (1-8). There are three classes of RNR, which are classified according to their different way to generate the transient thiyl radical (4, 7, 10 -14). In class I RNR enzymes a tyrosyl radical in a second subunit R2 is proposed to generate the transient thiyl radical near the bound substrate in subunit R1 by a long range proton coupled electron transfer process. The tyrosyl radical in R2 is generated by a reaction of a nearby diiron center with molecular oxygen (1-5, 15, 16). Class II RNRs utilize a cobalt cofactor, the vitamin B 12 derivative adenosylcobalamin, that interacts directly with the cysteine near the substrate to form the thiyl radical needed for the ribonucleotide reduction (4, 13, 14, 17). Class III RNRs consist of two subunits. A glycyl radical is generated in the...

show abstract

“…The tyrosyl radical in a second RNR, termed class Ib, which was found as the active aerobic enzyme in some bacteria, e.g., Mycobacterium tuberculosis, showed significantly different hf tensors of the b-protons of the tyrosyl side chain compared with the class Ia enzymes present in E. coli and mouse (Liu et al, 2000). Class Ib RNR has a very similar overall structure for R2 compared with class Ia from E. coli (Figure 1) or mouse, except for a greater distance between the tyrosyl radical and the diiron site.…”

Section: Structure and Protein Interactions Of The Tyrosyl Radical Inmentioning

confidence: 99%

“…However, in contrast to class Ia, class Ib lacks overall allosteric activity regulation (Eklund et al, 2001). The difference in hf tensors of the b-protons between class Ia and class Ib was attributed to geometrically different side-chain orientations (see Figure 3; Liu et al, 2000;Eklund et al, 2001;Lendzian, 2005) largely determined by protein constraints. Indeed, theoretical studies indicated that the side chain orientations of the tyrosyl radicals Y122…”

Section: Structure and Protein Interactions Of The Tyrosyl Radical Inmentioning

confidence: 99%

“…Using experimental and theoretical studies, this difference was traced back to polar interactions, in particular those that involve hydrogen bonding to the oxygen attached to the tyrosine ring, as in mouse and yeast RNR Engströ m et al, 2000;Un et al, 2001 Dam et al, 1998;Bar et al, 2001). Interestingly, the tyrosyl radical in class Ib Mycobacterium tuberculosis was found to exhibit a heterogeneity showing stretched g x values between 2.0092 and 2.0080 and it was speculated that the enzyme may be activated by connecting the radical to the chain of hydrogen bonds (Liu et al, 2000).…”

Section: Structure and Protein Interactions Of The Tyrosyl Radical Inmentioning

confidence: 99%

See 1 more Smart Citation

Spectroscopic and theoretical approaches for studying radical reactions in class I ribonucleotide reductase

Bennati

Lendzian²,

Schmittel³

et al. 2005

Biological Chemistry

View full text Add to dashboard Cite

Ribonucleotide reductases (RNRs) catalyze the production of deoxyribonucleotides, which are essential for DNA synthesis and repair in all organisms. The three currently known classes of RNRs are postulated to utilize a similar mechanism for ribonucleotide reduction via a transient thiyl radical, but they differ in the way this radical is generated. Class I RNR, found in all eukaryotic organisms and in some eubacteria and viruses, employs a diferric iron center and a stable tyrosyl radical in a second protein subunit, R2, to drive thiyl radical generation near the substrate binding site in subunit R1. From extensive experimental and theoretical research during the last decades, a general mechanistic model for class I RNR has emerged, showing three major mechanistic steps: generation of the tyrosyl radical by the diiron center in subunit R2, radical transfer to generate the proposed thiyl radical near the substrate bound in subunit R1, and finally catalytic reduction of the bound ribonucleotide. Amino acid-or substrate-derived radicals are involved in all three major reactions. This article summarizes the present mechanistic picture of class I RNR and highlights experimental and theoretical approaches that have contributed to our current understanding of this important class of radical enzymes.

show abstract

Heterogeneity of the Local Electrostatic Environment of the Tyrosyl Radical in Mycobacterium tuberculosis Ribonucleotide Reductase Observed by High-Field Electron Paramagnetic Resonance

Cited by 28 publications

References 37 publications

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Orientation of the Tyrosyl Radical inSalmonella typhimuriumClass Ib Ribonucleotide Reductase Determined by High Field EPR of R2F Single Crystals

Spectroscopic and theoretical approaches for studying radical reactions in class I ribonucleotide reductase

Contact Info

Product

Resources

About