Mechanism of Assembly of the Tyrosyl Radical-Dinuclear Iron Cluster Cofactor of Ribonucleotide Reductase

Bollinger, J. Martin; Edmondson, Dale E.; Huynh, Boi Hanh; Filley, Jonathan; Norton, Jack R.; Stubbe, JoAnne

doi:10.1126/science.1650033

Cited by 368 publications

(513 citation statements)

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“…Similarly, Mn(II) ions have been used to probe the mechanism of metal ion incorporation in the homodimeric diiron-dependent RNR [83][84][85][86]. Hendrich and coworkers used CW EPR data acquired at X-and Q-band frequencies to show that binding of a single Mn(II) ion to one of the domains of apo-RNR induced a significant allosteric event that prevented binding of an additional equivalent of Mn(II) to the other domain.…”

Section: Past Studiesmentioning

confidence: 99%

Multifrequency pulsed EPR studies of biologically relevant manganese(II) complexes

et al. 2007

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Electron paramagnetic resonance studies at multiple frequencies (MF EPR) can provide detailed electronic structure descriptions of unpaired electrons in organic radicals, inorganic complexes, and metalloenzymes. Analysis of these properties aids in the assignment of the chemical environment surrounding the paramagnet and provides mechanistic insight into the chemical reactions in which these systems take part. Herein, we present results from pulsed EPR studies performed at three different frequencies (9, 31, and 130 GHz) on [Mn(II)(H 2 O) 6 ] 2+ , Mn(II) adducts with the nucleotides ATP and GMP, and the Mn(II)-bound form of the hammerhead ribozyme (MnHH). Through line shape analysis and interpretation of the zero-field splitting values derived from successful simulations of the corresponding continuous-wave and field-swept echodetected spectra, these data are used to exemplify the ability of the MF EPR approach in distinguishing the nature of the first ligand sphere. A survey of recent results from pulsed EPR, as well as pulsed electron-nuclear double resonance and electron spin echo envelope modulation spectroscopic studies applied to Mn(II)-dependent systems, is also presented. Mn-Containing Biological SystemsManganese operates as a cofactor in numerous proteins, serving both catalytic and structural roles [1][2][3][4]. Many Mn-dependent enzymes take advantage o f the rich redox chemistry available to the metal, accessing the +2, +3, +4, and perhaps even the +5 oxidation states during their turnover. For example, Mn-superoxide dismutase (MnSOD), which detoxifies the cell of the superoxide radical , cycles between the Mn(II) and Mn(III) oxidation states via the ping-pong type mechanism shown below [5][6][7][8][9].(1a) (1b) Other examples of such mononuclear redox-active enzymes include the manganese peroxidase responsible for lignin degradation by white-rot fungus [10][11][12]; a unique Mndependent form of lipoxygenase [13][14][15][16]; oxalate decarboxylase [17,18]; as well as an extradiol catechol dioxygenase [19][20][21]. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptIn order to help minimize kinetic and thermodynamic penalties associated with electron transfer events and the execution of chemical reactions, two or more metal centers can be coupled together to provide active sites capable of conducting multiple electrons while still operating within a physiologically accessible range of reduction potentials [22]. Only three such examples of redox-active polynuclear Mn enzymes are known: Mn-catalase that disproportionates hydrogen peroxide [23][24][25][26]; a Mn form of ribonucleotide reductase (Mn-RNR) [27,28]; and, arguably the most recognized Mn-dependent enzyme, the photosynthetic core of green plants, algae, and certain cyanobacteria termed photosystem II (PSII) [29,30]. Through a series of photoinitiated electron transfer events, oxidizing equivalents are stored on the tetranuclear Mn core of PSII -the oxygen-evolving complex (OEC) -which then extracts four electr...

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Section: Past Studiesmentioning

confidence: 99%

Multifrequency pulsed EPR studies of biologically relevant manganese(II) complexes

et al. 2007

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“…Moreover, single (35) and multiple turnover (36,38) studies of what is generally believed to approximate the chain length of the physiological substrate, eicosanoic acid (EA), have indicated that oxidative decarboxylation, affording nonadecene, is the largely dominant reaction route (34). Combined 13 C substrate and H 2 18 O 2 isotopic labeling by our laboratory has shown that cleavage of the terminal fatty acid carboxylate does not lead to recovery of 18 …”

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confidence: 99%

“…Despite the ubiquity of this mechanism, in some metalloenzymes, a metal−oxo species is formed that is not ultimately destined for incorporation into a substrate. Some characterized examples include the nonheme dinuclear iron ribonucleotide reductase (RNR R2) (18,19) and mononuclear iron halogenase SyrB2 (20-22). The mechanisms for RNR and SyrB2 highlight the importance of quaternary structural elements, an extensive 35-Å proton-coupled electron transfer pathway in RNR (23), and extremely subtle (subangstrom) substrate positional tuning (SyrB2) (21,22), to enable efficient circumvention from the monooxygenation reaction coordinate.…”

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confidence: 99%

Catalytic strategy for carbon−carbon bond scission by the cytochrome P450 OleT

Grant

Mitchell

Makris

2016

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

102

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OleT is a cytochrome P450 that catalyzes the hydrogen peroxidedependent metabolism of C n chain-length fatty acids to synthesize C n-1 1-alkenes. The decarboxylation reaction provides a route for the production of drop-in hydrocarbon fuels from a renewable and abundant natural resource. This transformation is highly unusual for a P450, which typically uses an Fe 4+ −oxo intermediate known as compound I for the insertion of oxygen into organic substrates. OleT, previously shown to form compound I, catalyzes a different reaction. A large substrate kinetic isotope effect (≥8) for OleT compound I decay confirms that, like monooxygenation, alkene formation is initiated by substrate C−H bond abstraction. Rather than finalizing the reaction through rapid oxygen rebound, alkene synthesis proceeds through the formation of a reaction cycle intermediate with kinetics, optical properties, and reactivity indicative of an Fe 4+ −OH species, compound II. The direct observation of this intermediate, normally fleeting in hydroxylases, provides a rationale for the carbon−carbon scission reaction catalyzed by OleT.C ytochrome P450 (CYP) enzymes catalyze an extraordinary breadth of physiologically important oxidations for xenobiotic detoxification and specialized biosynthetic pathways (1, 2). The metabolic diversity of CYP enzymes originates from a sophisticated interplay of substrate molecular recognition with precise tuning of metal oxygen species formed at the enzyme active site. CYPs use a thiolate-ligated heme iron cofactor to activate molecular oxygen and produce short-lived ferric superoxo (3-5), ferric (hydro)peroxo (6-8), and ferryl (9, 10) intermediates. Coordinated efforts over several decades have resulted in isolation of each intermediate, including recent characterization of the principal oxidant thought to be responsible for the vast majority of P450 oxidations, the Fe 4+ −oxo pi−cation radical species commonly referred to as compound I (or P450-I) (10).The archetypal P450 hydroxylation involves C−H bond abstraction by P450-I and ensuing rapid oxygen insertion through a recombination process termed "oxygen rebound" (11, 12). The hydrogen abstraction/rebound mechanism describes the strategy used for most P450 transformations, and is thought to describe catalysis by numerous metal-dependent oxygenases and synthetic bio-inspired metal−oxo complexes (e.g., refs. 13-17). Despite the ubiquity of this mechanism, in some metalloenzymes, a metal−oxo species is formed that is not ultimately destined for incorporation into a substrate. Some characterized examples include the nonheme dinuclear iron ribonucleotide reductase (RNR R2) (18,19) and mononuclear iron halogenase SyrB2 (20-22). The mechanisms for RNR and SyrB2 highlight the importance of quaternary structural elements, an extensive 35-Å proton-coupled electron transfer pathway in RNR (23), and extremely subtle (subangstrom) substrate positional tuning (SyrB2) (21,22), to enable efficient circumvention from the monooxygenation reaction coordinate.P450-I has also been link...

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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.…”

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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-Dinuclear Iron Cluster Cofactor of Ribonucleotide Reductase

Cited by 368 publications

References 30 publications

Multifrequency pulsed EPR studies of biologically relevant manganese(II) complexes

Multifrequency pulsed EPR studies of biologically relevant manganese(II) complexes

Catalytic strategy for carbon−carbon bond scission by the cytochrome P450 OleT

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

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