Stable Tyrosyl Radical Research Articles

The Crystal Structure of an Azide Complex of the Diferrous R2 Subunit of Ribonucleotide Reductase Displays a Novel Carboxylate Shift with Important Mechanistic Implications for Diiron-Catalyzed Oxygen Activation

The dinuclear Fe-center in the R2 protein of ribonucleotide reductase catalyzes oxygen activation chemistry leading to generation of the essential stable tyrosyl radical. Related oxygen reactions occur in several other diiron-containing enzyme systems where highly oxidative reaction intermediates are required to activate the substrates. Two such examples are methane monooxygenase and Δ9 stearoyl-acyl carrier protein desaturase where oxygen activation takes place at Fe-centers whose structures are similar to the Fe-center in R2. In an attempt to structurally characterize the nature of the dioxygen cleavage reaction performed by these proteins we have determined the crystal structures of two different forms of the diferrous R2 protein in the presence of azide, a potential Fe ligand. In crystals of the wt protein no azide binding was detected. The mutant protein F208A/Y122F has a larger hydrophobic pocket around the Fe-center and in the structure of this protein azide bind as an η1-terminal ligand to Fe2, the Fe ion farthest away from the tyrosine residue to be oxidized in the radical generation reaction. Glu 238, the Fe ligand most exposed into the hydrophobic pocket, coordinates the Fe-center in a novel μ-(η2,η1) bridging mode with one of the carboxylate oxygen atoms forming a bridge between the two iron ions and the other oxygen being coordinated to Fe2. Through this bridging the Fe−Fe distance is shortened to about 3.4 Å as compared to 3.9 Å for the structure of the reduced wt protein. On the basis of the novel carboxylate shift and recent data on the spectroscopic properties of the key intermediate X we propose a unique structure for intermediate X and a detailed mechanism for dioxygen cleavage. This mechanism suggests an asymmetric oxygen cleavage with a terminal oxo/hydroxo group as the major species responsible for substrate activation.

Kinetics of Transient Radicals in Escherichia coli Ribonucleotide Reductase: FORMATION OF A NEW TYROSYL RADICAL IN MUTANT PROTEIN R2

Reconstitution of the tyrosyl radical in ribonucleotide reductase protein R2 requires oxidation of a diferrous site by oxygen. The reaction involves one externally supplied electron in addition to the three electrons provided by oxidation of the Tyr-122 side chain and formation of the mu-oxo-bridged diferric site. Reconstitution of R2 protein Y122F, lacking the internal pathway involving Tyr-122, earlier identified two radical intermediates at Trp-107 and Trp-111 in the vicinity of the di-iron site, suggesting a novel internal transfer pathway (Sahlin, M., Lassmann, G., Pötsch, S., Sjöberg, B. -M., and Gräslund, A. (1995) J. Biol. Chem. 270, 12361-12372). Here, we report the construction of the double mutant W107Y/Y122F and its three-dimensional structure and demonstrate that the tyrosine Tyr-107 can harbor a transient, neutral radical (Tyr-107(.)). The Tyr-107(.) signal exhibits the hyperfine structure of a quintet with coupling constants of 1.3 mT for one beta-methylene proton and 0.75 mT for each of the 3 and 5 hydrogens of the phenyl ring. Rapid freeze quench kinetics of EPR-visible intermediates reveal a preferred radical transfer pathway via Trp-111, Glu-204, and Fe-2, followed by a proton coupled electron transfer through the pi-interaction of the aromatic rings of Trp-(Tyr-)107 and Trp-111. The kinetic pattern observed in W107Y/Y122F is considerably changed as compared with Y122F: the Trp-111(.) EPR signal has vanished, and the Tyr-107(.) has the same formation rate as does Trp-111(.) in Y122F. According to the proposed consecutive reaction, Trp-111(.) becomes very short lived and is no longer detectable because of the faster formation of Tyr-107(.). We conclude that the phenyl rings of Trp-111 and Tyr-107 form a better stacking complex so that the proton-coupled electron transfer is facilitated compared with the single mutant. Comparison with the formation kinetics of the stable tyrosyl radical in wild type R2 suggests that these protein-linked radicals are substitutes for the missing Tyr-122. However, in contrast to Tyr-122(.) these radicals lack a direct connection to the radical transfer pathway utilized during catalysis.

High Field EPR Studies of Mouse Ribonucleotide Reductase Indicate Hydrogen Bonding of the Tyrosyl Radical

Ribonucleotide reductase catalyzes by free radical chemistry the reduction of ribonucleotides to deoxyribonucleotides. The R2 protein of a class 1 ribonucleotide reductase contains a stable tyrosyl radical of neutral phenoxy character, which is necessary for normal enzymatic activity. Here we present the EPR spectra from the tyrosyl free radical in the R2 protein from mouse at 9.62, 115, and 245 GHz. We show that the g-value anisotropy of the mouse R2 radical, when precisely determined from high field EPR spectra, is similar to that of the hydrogen bonded dark stable YD middle dot tyrosyl radical of photosystem II and different from that of the Escherichia coli R2 radical. Because the g-value anisotropy is an important indicator of the hydrogen bonding status of the tyrosyl radical, this result suggests that the mouse R2 radical has its tyrosylate oxygen hydrogen bonded with a D2O exchangeable proton, whereas this hydrogen bond is absent in the E. coli enzyme. It is suggested that the observed proton may be derived from the tyrosine that will become a tyrosyl radical.

Two Conserved Tyrosine Residues in Protein R1 Participate in an Intermolecular Electron Transfer in Ribonucleotide Reductase

The enzyme ribonucleotide reductase consists of two nonidentical proteins, R1 and R2, which are each inactive alone. R1 contains the active site and R2 contains a stable tyrosyl radical essential for catalysis. The reduction of ribonucleotides is radical-based, and a long range electron transfer chain between the active site in R1 and the radical in R2 has been suggested. To find evidence for such an electron transfer chain in Escherichia coli ribonucleotide reductase, we converted two conserved tyrosines in R1 into phenylalanines by site-directed mutagenesis. The mutant proteins were shown to be enzymatically inactive. In addition, the mechanism-based inhibitor 2'-azido-2'-deoxy-CDP was incapable of scavenging the R2 radical, and no azido-CDP-derived radical intermediate was formed. We also show that the loss of enzymatic activity was not due to impaired R1-R2 complex formation or substrate binding. Based on these results, we predict that the two tyrosines, Tyr-730 and Tyr-731, are part of a hydrogen-bonded network that constitutes an electron transfer pathway in ribonucleotide reductase. It is demonstrated that there is no electron delocalization over these tyrosines in the resting wild-type complex.

Reconsideration of X, the Diiron Intermediate Formed during Cofactor Assembly in E. coli Ribonucleotide Reductase

The R2 subunit of Escherichia coli ribonucleotide reductase (RNR) contains a stable tyrosyl radical (•Y122) diferric cluster cofactor. Earlier studies on the cofactor assembly reaction detected a paramagnetic intermediate, X, that was found to be kinetically competent to oxidize Y122. Studies using rapid freeze-quench (RFQ) Mossbauer and EPR spectroscopies led to the proposal that X is comprised of two high spin ferric ions and a S = 1/2 ligand radical, mutually spin coupled to give a S = 1/2 ground state (Ravi, N.; Bollinger, J. M., Jr.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8007−8014). An extension of RFQ methodology to Q-band ENDOR spectroscopy using 57Fe has shown that one of the irons has a very nearly isotropic hyperfine tensor (A(FeA) = −[74.2(2), 72.2(2), 73.2(2)] MHz) as expected for FeIII, but that the other iron site displays considerable anisotropy (A(FeB) = +[27.5(2), 36.8(2), 36.8(2)] MHz), indicative of substantial FeIV character. Reanalysis of the Mossbaue...

The Stable Tyrosyl Radical in Mouse Ribonucleotide Reductase Is Not Essential for Enzymic Activity

The Stable Tyrosyl Radical in Mouse Ribonucleotide Reductase Is Not Essential for Enzymic Activity

Angular orientation of the stable tyrosyl radical within photosystem II by high-field 245-GHz electron paramagnetic resonance.

The 4 K 245-GHz/8.7-T electron paramagnetic resonance spectrum of the stable tyrosyl radical in photosystem II, known as TyrD., has been measured. Illumination at 200 K enhances the signal intensity of TyrD. by a factor of > 40 compared to the signal obtained from dark-adapted samples. This signal enhancement and the unusual line shape of the TyrD. resonance result from the magnetic dipolar coupling of the radical to the manganese cluster involved in oxygen evolution. The relative angular orientation of the manganese cluster with respect to TyrD. has been determined from line-shape analysis. The resonance arising from TyrD. in Tris-washed manganese-free photosystem II sample is also distorted. This effect probably originates from the influence of the nonheme iron on the spin relaxation of the tyrosyl radical. The relative angular orientation of the nonheme iron has also been determined. Oriented samples were used to determine the angular orientation of TyrD. with respect to the membrane plane. Combining angular data with published distances, we have constructed a three-dimensional picture of the relative positions of TyrD., the manganese cluster, and the nonheme iron. The data suggest a more symmetrical placement of the manganese relative to TyrD. and TyrZ, the tyrosine involved in electron transfer, than is usually assumed in current models of photosystem II.

Substitution of manganese for iron in ribonucleotide reductase from Escherichia coli. Spectroscopic and crystallographic characterization.

Each polypeptide chain of protein R2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a stable tyrosyl radical and two antiferromagnetically coupled oxo-bridged ferric ions. A refined structure of R2 has been recently obtained. R2 can be converted into apoR2 by chelating out the metal cofactor and scavenging the radical. This study shows that apoR2 has a very strong affinity for four stable Mn2+ ions. The manganese-containing form of R2, named Mn-R2, has been studied by EPR spectroscopy and x-ray crystallography. It contains two binuclear manganese clusters in which the two manganese ions occupy the natural iron-binding sites and are only bridged by carboxylates from glutamates 115 and 238. This in turn explains why the spin-exchange interaction between the two ions is very weak and why Mn-R2 is EPR active. Mn-R2 could provide a model for the native diferrous form of protein R2, and a detailed molecular mechanism for the reduction of the iron center of protein R2 is proposed.