Mechanism of Assembly of the Tyrosyl Radical-Diiron(III) Cofactor of E. Coli Ribonucleotide Reductase: 1. Moessbauer Characterization of the Diferric Radical Precursor

Ravi, Natarajan; Bollinger, J. Martin; Huynh, Boi Hanh; Stubbe, JoAnne; Edmondson, Dale E.

doi:10.1021/ja00097a007

Cited by 211 publications

(315 citation statements)

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“…Theoretical VTVH MCD saturation magnetization data were constructed as published previously (29). Mössbauer spectra were recorded using spectrometers described previously (30). The zero velocity refers to the centroid of the room temperature spectra of metallic iron foil.…”

Section: Methodsmentioning

confidence: 99%

Characterization of MOCS1A, an Oxygen-sensitive Iron-Sulfur Protein Involved in Human Molybdenum Cofactor Biosynthesis

Hänzelmann

Hernandez

Menzel

et al. 2004

Journal of Biological Chemistry

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138

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The human proteins MOCS1A and MOCS1B catalyze the conversion of a guanosine derivative to precursor Z during molybdenum cofactor biosynthesis. MOCS1A shares homology with S-adenosylmethionine (AdoMet)-dependent radical enzymes, which catalyze the formation of protein and/or substrate radicals by reductive cleavage of AdoMet through a [4Fe-4S] cluster. Sequence analysis of MOCS1A showed two highly conserved cysteine motifs, one near the N terminus and one near the C terminus. MOCS1A was heterologously expressed in Escherichia coli and purified under aerobic and anaerobic conditions. Individual mutations of the conserved cysteines to serine revealed that all are essential for synthesis of precursor Z in vivo. The molybdenum cofactor in eukaryotic molybdoenzymes consists of a mononuclear molybdenum coordinated by the dithiolene moiety of a tricyclic pyranopterin, termed molybdopterin (MPT) 1 (1). In humans, defects in molybdenum cofactor biosynthesis lead to the pleiotropic loss of the molybdoenzymes sulfite oxidase, aldehyde oxidase, and xanthine dehydrogenase (2, 3). Affected patients usually die shortly after birth and show neurological abnormalities, such as attenuated growth of the brain, untreatable seizures, and dislocated ocular lenses (4). The first step during human molybdenum cofactor biosynthesis is catalyzed by MOCS1A and MOCS1B, leading to the synthesis of precursor Z, an oxygen-sensitive 6-alkyl pterin with a cyclic phosphate, from a guanosine derivative, most likely 5Ј-GTP (5-7).Analogous to other pteridine biosynthetic pathways, synthesis of precursor Z has been proposed to occur via a GTP cyclohydrolase-like reaction mechanism (5, 6). In contrast to these pathways, the C-8 atom of 5Ј-GTP is not released as formate but is retained and incorporated in a rearrangement reaction as the first carbon atom of the precursor Z side chain. In the second step of molybdenum cofactor biosynthesis catalyzed by MOCS3 (8) and MPT synthase (MOCS2) precursor Z is converted into MPT (9 -11). Finally molybdenum is incorporated into MPT by the multifunctional protein gephyrin (12, 13).MOCS1A contains two highly conserved cysteine motifs ( Fig. 1) proposed to be involved in iron-sulfur (FeS) cluster binding (14, 15), one is located near the N terminus (consensus sequence CX 3 CX 2 C where X denotes any amino acid), and one is near the C terminus (consensus sequence CX 2 CX 13 C). Several mutations identified in molybdenum cofactor deficiency patients are located in these conserved cysteine motifs indicating their functional importance for protein activity (2, 3). Based on sequence similarities to proteins such as biotin synthase, pyruvate formate-lyase-activating enzyme, and anaerobic ribonucleotide reductase-activating enzyme, MOCS1A has been classified as a member of the superfamily of S-adenosylmethionine (AdoMet)-dependent radical enzymes (16). In this class of enzymes, AdoMet serves as the free radical initiator and undergoes cleavage to methionine and a 5Ј-deoxyadenosyl radical that in turn propagates radical for...

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Section: Methodsmentioning

confidence: 99%

Characterization of MOCS1A, an Oxygen-sensitive Iron-Sulfur Protein Involved in Human Molybdenum Cofactor Biosynthesis

Hänzelmann

Hernandez

Menzel

et al. 2004

Journal of Biological Chemistry

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138

124

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“…'X' has been investigated extensively using a variety of spectroscopic methods. Early investigations using Mö ssbauer spectroscopy assigned 'X' as a strongly coupled diferric iron-radical center (Bollinger et al, 1994b;Ravi et al, 1994). In a later, combined 35-GHz ENDOR and Mö ssbauer study, it was shown that 'X' is better described as a Fe III Fe IV state (Sturgeon et al, 1996).…”

Section: Functional Transient Diiron States and Radicals In The Activmentioning

confidence: 99%

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

Bennati

Lendzian²,

Schmittel³

et al. 2005

Biological Chemistry

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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.

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“…The function of the metal site appears to be the generation and stabilization of the radical (7)(8)(9)(10). Protein R2 having the tyrosyl radical reduced to normal tyrosine is called Met-R2 (R2 Met ) and is enzymatically inactive.…”

mentioning

confidence: 99%

Redox Studies of Subunit Interactivity in Aerobic Ribonucleotide Reductase from Escherichia coli

Zlateva

Quaroni²,

Que

et al. 2004

Journal of Biological Chemistry

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Ribonucleotide reductase is a heterodimeric (␣ 2 ␤ 2 ) allosteric enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides, an essential step in DNA biosynthesis and repair. In the enzymatically active form aerobic Escherichia coli ribonucleotide reductase is a complex of homodimeric R1 and R2 proteins. We use electrochemical studies of the dinuclear center to clarify the interplay of subunit interaction, the binding of allosteric effectors and substrate selectivity. Our studies show for the first time that electrochemical reduction of active R2 generates a distinct Met form of the diiron cluster, with a midpoint potential (؊163 ؎ 3 mV) different from that of R2 Met produced by hydroxyurea (؊115 ؎ 2 mV). The redox potentials of both Met forms experience negative shifts when measured in the presence of R1, becoming ؊223 ؎ 6 and ؊226 ؎ 3 mV, respectively, demonstrating that R1-triggered conformational changes favor one configuration of the diiron cluster. We show that the association of a substrate analog and specificity effector (dGDP/dTTP or GMP/dTTP) with R1 regulates the redox properties of the diiron centers in R2. Their midpoint potential in the complex shifts to ؊192 ؎ 2 mV for dGDP/dTTP and to ؊203 ؎ 3 mV for GMP/dTTP. In contrast, reduction potential measurements show that the diiron cluster is not affected by ATP (0.35-1.45 mM) and dATP (0.3-0.6 mM) binding to R1. Binding of these effectors to the R1-R2 complex does not perturb the normal docking modes between R1 and R2 as similar redox shifts are observed for ATP or dATP associated with the R1-R2 complex.Ribonucleotide reductases (RNRs) 1 catalyze the reduction of the four canonical ribonucleotides to the corresponding deoxyribonucleotides in all organisms, thus providing the precursors for DNA synthesis. Allosteric regulation ensures a balanced dNTP pool needed for DNA replication and repair. At present, three major classes of RNRs have been identified that differ in their protein structure and in the cofactors essential for catalysis (1). Despite these differences, all RNRs catalyze the reduction of ribonucleotides by similar radical-based mechanisms, including formation of a transient thiyl radical at the active site that in turn produces a transient 3Ј-substrate radical (2). In class I enzymes, it is proposed that the thiyl radical within the R1 subunit is formed during each catalytic cycle via long range electron/proton transfer from a stable tyrosyl radical located in the R2 subunit (1, 2).The enzymatic activity of aerobic Escherichia coli RNR, which belongs to the class I RNR like the mammalian enzyme, depends on the formation of the complex between the two different proteins, R1 (␣ 2 ) and R2 (␤ 2 ), and is regulated by the binding of dNTPs and ATP, which act as allosteric effectors. The R1 homodimer carries two types of regulatory sites in addition to the catalytic binding site. Overall activity is stimulated by ATP and inhibited by dATP through the binding of these effectors to the activity site (3). The regulation of su...

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Mechanism of Assembly of the Tyrosyl Radical-Diiron(III) Cofactor of E. Coli Ribonucleotide Reductase: 1. Moessbauer Characterization of the Diferric Radical Precursor

Cited by 211 publications

References 8 publications

Characterization of MOCS1A, an Oxygen-sensitive Iron-Sulfur Protein Involved in Human Molybdenum Cofactor Biosynthesis

Characterization of MOCS1A, an Oxygen-sensitive Iron-Sulfur Protein Involved in Human Molybdenum Cofactor Biosynthesis

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

Redox Studies of Subunit Interactivity in Aerobic Ribonucleotide Reductase from Escherichia coli

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