Cell proliferation and gill morphology in anoxic crucian carp

Is DNA replication/cell proliferation in vertebrates possible during anoxia? The oxygen dependence of ribonucleotide reductase (RNR) could lead to a stop in DNA synthesis, thereby making anoxic DNA replication impossible. We have studied this question in an anoxia-tolerant vertebrate, the crucian carp (Carassius carassius), by examining 5'-bromo-2'-deoxyuridine incorporation and proliferating cell nuclear antigen levels in the gills, intestinal crypts, and liver. We exposed crucian carp to 1 and 7 days of anoxia followed by 7 days of reoxygenation. There was a reduced incidence of S-phase cells (from 12.2 to 5.0%) in gills during anoxia, which coincided with a concomitant increase of G(0) cells. Anoxia also decreased the number of S-phase cells in intestine (from 8.1 to 1.8%). No change in the fraction of S-phase cells ( approximately 1%) in liver was found. Thus new S-phase cells after 7 days of anoxia were present in all tissues, revealing a considerable rate of DNA synthesis. Subsequently, the oxygen-dependent subunit of crucian carp RNR (RNRR2) was cloned. We found no differences in amino acids involved in radical generation and availability of the iron center compared with mouse, which could have explained reduced oxygen dependence. Furthermore, the amount of RNRR2 mRNA in gills did not decrease throughout anoxia exposure. These results indicate that crucian carp is able to sustain some cell proliferation in anoxia, possibly because RNRR2 retains its tyrosyl radical in anoxia, and that the replication machinery is still maintained. Although hypoxia triggers a 7.5-fold increase of respiratory surface area in crucian carp, this response was not triggered in anoxia.     

Circular dichroism and magnetic circular dichroism studies of the active site of p53R2 from human and mouse: iron binding and nature of the biferrous site relative to other ribonucleotide reductases

Ribonucleotide reductases (RNR) catalyze the rate-limiting step in the synthesis of deoxyribonucleotides from the corresponding ribonucleotides in the synthesis of DNA. Class I RNR has two subunits: R1 with the substrate binding and active site and R2 with a stable tyrosyl radical and diiron cluster. Biferrous R2 reacts with oxygen to form the tyrosyl radical needed for enzymatic activity. A novel R2 form, p53R2, is a 351-amino acid protein induced by the "tumor suppressor gene" p53. p53R2 has been studied using a combination of circular dichroism, magnetic circular dichroism, variable-temperature variable-field MCD, and EPR spectroscopies. The active site of biferrous p53R2 in both the human (hp53R2) and mouse (mp53R2) forms is found to have one five-coordinate and one four-coordinate iron, which are weakly antiferromagnetically coupled through mu-1,3-carboxylate bridges. These spectroscopic data are very similar to those of Escherichia coli R2, and mouse R2, with a stronger resemblance to data of the former. Titrations of apo-hp53R2 and apo-mp53R2 with Fe(II) were pursued for the purpose of comparing their metal binding affinities to those of other R2s. Both p53R2s were found to have a high affinity for Fe(II), which is different from that of mouse R2 and may reflect differences in the regulation of enzymatic activity, as p53R2 is mainly triggered during DNA repair. The difference in ferrous affinity between mammalian R2 and p53R2 suggests the possibility of specific inhibition of DNA precursor synthesis during cell division.     

Structure and interactions of amino acid radicals in class I ribonucleotide reductase studied by ENDOR and high-field EPR spectroscopy

This short review compiles high-field electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies on different intermediate amino acid radicals, which emerge in wild-type and mutant class I ribonucleotide reductase (RNR) both in the reaction of protein subunit R2 with molecular oxygen, which generates the essential tyrosyl radical, and in the catalytic reaction, which involves a radical transfer between subunits R2 and R1. Recent examples are presented, how different amino acid radicals (tyrosyl, tryptophan, and different cysteine-based radicals) were identified, assigned to a specific residue, and their interactions, in particular hydrogen bonding, were investigated using high-field EPR and ENDOR spectroscopy. Thereby, unexpected diiron-radical centers, which emerge in mutants of R2 with changed iron coordination, and an important catalytic cysteine-based intermediate in the substrate turnover reaction in R1 were identified and characterized. Experiments on the essential tyrosyl radical in R2 single crystals revealed the so far unknown conformational changes induced by formation of the radical. Interesting structural differences between the tyrosyl radicals of class Ia and Ib enzymes were revealed. Recently accurate distances between the tyrosyl radicals in the protein dimer R2 could be determined using pulsed electron-electron double resonance (PELDOR), providing a new tool for docking studies of protein subunits. These studies show that high-field EPR and ENDOR are important tools for the identification and investigation of radical intermediates, which contributed significantly to the current understanding of the reaction mechanism of class I RNR.     

Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating cells

Recently, a homologue of the small subunit of mammalian ribonucleotide reductase (RNR) was discovered, called p53R2. Unlike the well characterized S phase-specific RNR R2 protein, the new form was induced in response to DNA damage by the p53 protein. Because the R2 protein is specifically degraded in late mitosis and absent in G0/G1 cells, the induction of the p53R2 protein may explain how resting cells can obtain deoxyribonucleotides for DNA repair. However, no direct demonstration of RNR activity of the p53R2 protein was presented and furthermore, no corresponding RNR large subunit was identified. In this study we show that recombinant, highly purified human and mouse p53R2 proteins contain an iron-tyrosyl free radical center, and both proteins form an active RNR complex with the human and mouse R1 proteins. UV irradiation of serum-starved, G0/G1-enriched mouse fibroblasts, stably transformed with an R1 promoter-luciferase reporter gene construct, caused a 3-fold increase in luciferase activity 24 h after irradiation, paralleled by an increase in the levels of R1 protein. Taken together, our data indicate that the R1 protein can function as the normal partner of the p53R2 protein and that an R1-p53R2 complex can supply resting cells with deoxyribonucleotides for DNA repair.     

Structure and interactions of amino acid radicals in class I ribonucleotide reductase studied by ENDOR and high-field EPR spectroscopy

This short review compiles high-field electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies on different intermediate amino acid radicals, which emerge in wild-type and mutant class I ribonucleotide reductase (RNR) both in the reaction of protein subunit R2 with molecular oxygen, which generates the essential tyrosyl radical, and in the catalytic reaction, which involves a radical transfer between subunits R2 and R1. Recent examples are presented, how different amino acid radicals (tyrosyl, tryptophan, and different cysteine-based radicals) were identified, assigned to a specific residue, and their interactions, in particular hydrogen bonding, were investigated using high-field EPR and ENDOR spectroscopy. Thereby, unexpected diiron-radical centers, which emerge in mutants of R2 with changed iron coordination, and an important catalytic cysteine-based intermediate in the substrate turnover reaction in R1 were identified and characterized. Experiments on the essential tyrosyl radical in R2 single crystals revealed the so far unknown conformational changes induced by formation of the radical. Interesting structural differences between the tyrosyl radicals of class Ia and Ib enzymes were revealed. Recently accurate distances between the tyrosyl radicals in the protein dimer R2 could be determined using pulsed electron-electron double resonance (PELDOR), providing a new tool for docking studies of protein subunits. These studies show that high-field EPR and ENDOR are important tools for the identification and investigation of radical intermediates, which contributed significantly to the current understanding of the reaction mechanism of class I RNR.     

Regulation of mammalian ribonucleotide reduction and dNTP pools after DNA damage and in resting cells

Ribonucleotide reductase (RNR) provides the cell with a balanced supply of deoxyribonucleoside triphosphates (dNTP) for DNA synthesis. In budding yeast DNA damage leads to an up-regulation of RNR activity and an increase in dNTP pools, which are essential for survival. Mammalian cells contain three non-identical subunits of RNR; that is, one homodimeric large subunit, R1, carrying the catalytic site and two variants of the homodimeric small subunit, R2 and the p53-inducible p53R2, each containing a tyrosyl free radical essential for catalysis. S-phase-specific DNA replication is supported by an RNR consisting of the R1 and R2 subunits. In contrast, DNA damage induces expression of the R1 and the p53R2 subunits. We now show that neither logarithmically growing nor G(o)/G1-synchronized mammalian cells show any major increase in their dNTP pools after DNA damage. However, non-dividing fibroblasts expressing the p53R2 protein, but not the R2 protein, have reduced dNTP levels if exposed to the RNR-specific inhibitor hydroxyurea, strongly indicating that there is ribonucleotide reduction in resting cells. The slow, 4-fold increase in p53R2 protein expression after DNA damage results in a less than 2-fold increase in the dNTP pools in G(o)/G1 cells, where the pools are about 5% that of the size of the pools in S-phase cells. Our results emphasize the importance of the low constitutive levels of p53R2 in mammalian cells, which together with low levels of R1 protein may be essential for the supply of dNTPs for basal levels of DNA repair and mitochondrial DNA synthesis in G(o)/G1 cells.     

Differential regulation of AMP-activated kinase and AKT kinase in response to oxygen availability in crucian carp (Carassius carassius)

We investigated whether two kinases critical for survival during periods of energy deficiency in anoxia-intolerant mammalian species, AMP-activated kinase (AMPK), and protein kinase B (AKT), are equally important for hypoxic/anoxic survival in the extremely anoxia-tolerant crucian carp (Carassius carassius). We report that phosphorylation of AMPK and AKT in heart and brain showed small changes after 10 days of severe hypoxia (0.3 mg O2/l at 9 degrees C). In contrast, anoxia exposure (0.01 mg O2/l at 8 degrees C) substantially increased AMPK phosphorylation but decreased AKT phosphorylation in carp heart and brain, indicating activation of AMPK and deactivation of AKT. In agreement, blocking the activity of AMPK in anoxic fish in vivo with 20 mg/kg Compound C resulted in an elevated metabolic rate (as indicated by increased ethanol production) and tended to reduce energy charge. This is the first in vivo experiment with Compound C in a nonmammalian vertebrate, and it appears that AMPK plays a role in mediating anoxic metabolic depression in crucian carp. Real-time RT-PCR analysis of the investigated AMPK subunit revealed that the most likely composition of subunits in the carp heart is alpha2, beta1B, gamma2a, whereas a more even expression of subunits was found in the brain. In the heart, expression of the regulatory gamma2-subunit increased in the heart during anoxia. In the brain, expression of the alpha1-, alpha2-, and gamma1-subunits decreased with anoxia exposure, but expression of the gamma2-subunit remained constant. Combined, our findings suggest that AMPK and AKT may play important, but opposing roles for hypoxic/anoxic survival in the anoxia-tolerant crucian carp.     

A stable FeIII-FeIV replacement of tyrosyl radical in a class I ribonucleotide reductase

Ribonucleotide reductase (RNR) of Chlamydia trachomatis is a class I RNR enzyme composed of two homodimeric components, proteins R1 and R2. In class I RNR, R1 has the substrate binding site, whereas R2 has a diferric site and normally in its active form a stable tyrosyl free radical. C. trachomatis RNR is unusual, because its R2 component has a phenylalanine in the place of the radical carrier tyrosine. Replacing the tyrosyl radical, a paramagnetic Fe(III)-Fe(IV) species (species X, normally a transient intermediate in the process leading to radical formation) may provide the oxidation equivalent needed to start the catalytic process via long range electron transfer from the active site in R1. Here EPR spectroscopy shows that in C. trachomatis RNR, species X can become essentially stable when formed in a complete RNR (R1/R2/substrate) complex, adding further weight to the possible role of this species X in the catalytic reaction.     

The active form of the R2F protein of class Ib ribonucleotide reductase from Corynebacterium ammoniagenes is a diferric protein

Corynebacterium ammoniagenes contains a ribonucleotide reductase (RNR) of the class Ib type. The small subunit (R2F) of the enzyme has been proposed to contain a manganese center instead of the dinuclear iron center, which in other class I RNRs is adjacent to the essential tyrosyl radical. The nrdF gene of C. ammoniagenes, coding for the R2F component, was cloned in an inducible Escherichia coli expression vector and overproduced under three different conditions: in manganese-supplemented medium, in iron-supplemented medium, and in medium without addition of metal ions. A prominent typical tyrosyl radical EPR signal was observed in cells grown in rich medium. Iron-supplemented medium enhanced the amount of tyrosyl radical, whereas cells grown in manganese-supplemented medium had no such radical. In highly purified R2F protein, enzyme activity was found to correlate with tyrosyl radical content, which in turn correlated with iron content. Similar results were obtained for the R2F protein of Salmonella typhimurium class Ib RNR. The UV-visible spectrum of the C. ammoniagenes R2F radical has a sharp 408-nm band. Its EPR signal at g = 2.005 is identical to the signal of S. typhimurium R2F and has a doublet with a splitting of 0.9 millitesla (mT), with additional hyperfine splittings of 0.7 mT. According to X-band EPR at 77-95 K, the inactive manganese form of the C. ammoniagenes R2F has a coupled dinuclear Mn(II) center. Different attempts to chemically oxidize Mn-R2F showed no relation between oxidized manganese and tyrosyl radical formation. Collectively, these results demonstrate that enzymatically active C. ammoniagenes RNR is a generic class Ib enzyme, with a tyrosyl radical and a diferric metal cofactor.     

EPR studies on a stable sulfinyl radical observed in the iron-oxygen-reconstituted Y177F/I263C protein R2 double mutant of ribonucleotide reductase from mouse

Ribonucleotide reductase (RNR) catalyzes the biosynthesis of deoxyribonucleotides. The active enzyme contains a diiron center and a tyrosyl free radical required for enzyme activity. The radical is located at Y177 in the R2 protein of mouse RNR. The radical is formed concomitantly with the mu-oxo-bridged diferric center in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. EPR at 9.6 and 285 GHz was used to investigate the reconstitution reaction in the double-mutant Y177F/I263C of mouse protein R2. The aim was to produce a protein-linked radical derived from the Cys residue in the mutant protein to investigate its formation and characteristics. The mutation Y177F hinders normal radical formation at Y177, and the I263C mutation places a Cys residue at the same distance from the iron center as Y177 in the native protein. In the reconstitution reaction, we observed small amounts of a transient radical with a probable assignment to a peroxy radical, followed by a stable sulfinyl radical, most likely located on C263. The unusual radical stability may be explained by the hydrophobic surroundings of C263, which resemble the hydrophobic pocket surrounding Y177 in native protein R2. The observation of a sulfinyl radical in RNR strengthens the relationship between RNR and another free radical enzyme, pyruvate formate-lyase, where a similar relatively stable sulfinyl radical has been observed in a mutant. Sulfinyl radicals may possibly be considered as stabilized forms of very short-lived thiyl radicals, proposed to be important intermediates in the radical chemistry of RNR.     

Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g ₁-value of 2.0090 for the tyrosyl radical was extracted. This g ₁-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν_7a = 1500 cm⁻¹) was found to be insensitive to deuterium-oxide exchange. Additionally, the ¹⁸O-sensitive Fe-O-Fe symmetric stretching (483 cm⁻¹) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g ₁-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053–33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher activity.     

Dramatic increase of nitrite levels in hearts of anoxia-exposed crucian carp supporting a role in cardioprotection

Nitrite (NO(2)(-)) functions as an important nitric oxide (NO) donor under hypoxic conditions. Both nitrite and NO have been found to protect the mammalian heart and other tissues against ischemia (anoxia)-reoxygenation injury by interacting with mitochondrial electron transport complexes and limiting the generation of reactive oxygen species upon reoxygenation. The crucian carp naturally survives extended periods without oxygen in an active state, which has made it a model for studying how evolution has solved the problems of anoxic survival. We investigated the role of nitrite and NO in the anoxia tolerance of this fish by measuring NO metabolites in normoxic, anoxic, and reoxygenated crucian carp. We also cloned and sequenced crucian carp NO synthase variants and quantified their mRNA levels in several tissues in normoxia and anoxia. Despite falling levels of blood plasma nitrite, the crucian carp showed massive increases in nitrite, S-nitrosothiols (SNO), and iron-nitrosyl (FeNO) compounds in anoxic heart tissue. NO(2)(-) levels were maintained in anoxic brain, liver, and gill tissues, whereas SNO and FeNO increased in a tissue-specific manner. Reoxygenation reestablished normoxic values. We conclude that NO(2)(-) is shifted into the tissues where it acts as NO donor during anoxia, inducing cytoprotection under anoxia/reoxygenation. This can be especially important in the crucian carp heart, which maintains output in anoxia. NO(2)(-) is currently tested as a therapeutic drug against reperfusion damage of ischemic hearts, and the present study provides evolutionary precedent for such an approach.     

Orientation of the tyrosyl radical in Salmonella typhimurium class Ib ribonucleotide reductase determined by high field EPR of R2F single crystals

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*. 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* 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* in E. coli R2. Presence of the substrate binding subunit R1E did not change the EPR spectra of Tyr-105*, 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.     

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

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.     

In vivo assay for low-activity mutant forms of Escherichia coli ribonucleotide reductase

Ribonucleotide reductase (RNR) catalyzes the essential production of deoxyribonucleotides in all living cells. In this study we have established a sensitive in vivo assay to study the activity of RNR in aerobic Escherichia coli cells. The method is based on the complementation of a chromosomally encoded nonfunctional RNR with plasmid-encoded RNR. This assay can be used to determine in vivo activity of RNR mutants with activities beyond the detection limits of traditional in vitro assays. E. coli RNR is composed of two homodimeric proteins, R1 and R2. The R2 protein contains a stable tyrosyl radical essential for the catalysis that takes place at the R1 active site. The three-dimensional structures of both proteins, phylogenetic studies, and site-directed mutagenesis experiments show that the radical is transferred from the R2 protein to the active site in the R1 protein via a radical transfer pathway composed of at least nine conserved amino acid residues. Using the new assay we determined the in vivo activity of mutants affecting the radical transfer pathway in RNR and identified some residual radical transfer activity in two mutant R2 constructs (D237N and W48Y) that had previously been classified as negative for enzyme activity. In addition, we show that the R2 mutant Y356W is completely inactive, in sharp contrast to what has previously been observed for the corresponding mutation in the mouse R2 enzyme.     

HF-EPR, Raman, UV/VIS light spectroscopic, and DFT studies of the ribonucleotide reductase R2 tyrosyl radical from Epstein-Barr virus

Epstein-Barr virus (EBV) belongs to the gamma subfamily of herpes viruses, among the most common pathogenic viruses in humans worldwide. The viral ribonucleotide reductase small subunit (RNR R2) is involved in the biosynthesis of nucleotides, the DNA precursors necessary for viral replication, and is an important drug target for EBV. RNR R2 generates a stable tyrosyl radical required for enzymatic turnover. Here, the electronic and magnetic properties of the tyrosyl radical in EBV R2 have been determined by X-band and high-field/high-frequency electron paramagnetic resonance (EPR) spectroscopy recorded at cryogenic temperatures. The radical exhibits an unusually low g₁-tensor component at 2.0080, indicative of a positive charge in the vicinity of the radical. Consistent with these EPR results a relatively high C-O stretching frequency associated with the phenoxyl radical (at 1508 cm⁻¹) is observed with resonance Raman spectroscopy. In contrast to mouse R2, EBV R2 does not show a deuterium shift in the resonance Raman spectra. Thus, the presence of a water molecule as a hydrogen bond donor moiety could not be identified unequivocally. Theoretical simulations showed that a water molecule placed at a distance of 2.6 Å from the tyrosyl-oxygen does not result in a detectable deuterium shift in the calculated Raman spectra. UV/VIS light spectroscopic studies with metal chelators and tyrosyl radical scavengers are consistent with a more accessible dimetal binding/radical site and a lower affinity for Fe²⁺ in EBV R2 than in Escherichia coli R2. Comparison with previous studies of RNR R2s from mouse, bacteria, and herpes viruses, demonstrates that finely tuned electronic properties of the radical exist within the same RNR R2 Ia class.     

Crystal structural studies of changes in the native dinuclear iron center of ribonucleotide reductase protein R2 from mouse

Class I ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleotides in mammals and many other organisms. The RNR subunit R2 contains a dinuclear iron center, which in its diferrous form spontaneously reacts with O2, forming a mu-oxo-bridged diferric cluster and a stable tyrosyl radical. Here, we present the first crystal structures of R2 from mouse with its native dinuclear iron center, both under reducing and oxidizing conditions. In one structure obtained under reducing conditions, the iron-bridging ligand Glu-267 adopts the mu-(eta1,eta2) coordination mode, which has previously been related to O2 activation, and an acetate ion from the soaking solution is observed where O2 has been proposed to bind the iron. The structure of mouse R2 under oxidizing conditions resembles the nonradical diferric R2 from Escherichia coli, with the exception of the coordination of water and Asp-139 to Fe1. There are also additional water molecules near the tyrosyl radical site, as suggested by previous spectroscopic studies. Since no crystal structure of the active radical form has been reported, we propose models for the movement of waters and/or tyrosyl radical site when diferric R2 is oxidized to the radical form, in agreement with our previous ENDOR study. Compared with E. coli R2, two conserved phenylalanine residues in the hydrophobic environment around the diiron center have opposing rotameric conformations, and the carboxylate ligands of the diiron center in mouse R2 appear more flexible. Together, this might contribute to the lower affinity and cooperative binding of iron in mouse R2.     

Intrinsic contractile properties of the crucian carp (Carassius carassius) heart during anoxic and acidotic stress

The crucian carp (Carassius carassius) seems unique among vertebrates in its ability to maintain cardiac performance during prolonged anoxia. We investigated whether this phenomenon arises in part from a myocardium tolerant to severe acidosis or because the anoxic crucian carp heart may not experience a severe extracellular acidosis due to the fish's ability to convert lactate to ethanol. Spontaneously contracting heart preparations from cold-acclimated (6-8°C) carp were exposed (at 6.5°C) to graded or ungraded levels of acidosis under normoxic or anoxic conditions and intrinsic contractile performance was assessed. Our results clearly show that the carp heart is tolerant of acidosis as long as oxygen is available. However, heart rate and contraction kinetics of anoxic hearts were severely impaired when extracellular pH was decreased below 7.4. Nevertheless, the crucian carp heart was capable of recovering intrinsic contractile performance upon reoxygenation regardless of the severity of the anoxic + acidotic insult. Finally, we show that increased adrenergic stimulation can ameliorate, to a degree, the negative effects of severe acidosis on the intrinsic contractile properties of the anoxic crucian carp heart. Combined, these findings indicate an avoidance of severe extracellular acidosis and adrenergic stimulation are two important factors protecting the intrinsic contractile properties of the crucian carp heart during prolonged anoxia, and thus likely facilitate the ability of the anoxic crucian carp to maintain cardiac pumping.     

Rnr4p, a novel ribonucleotide reductase small-subunit protein.

Ribonucleotide reductases catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. Eukaryotic ribonucleotide reductases are alpha2beta2 tetramers; each of the larger, alpha subunits possesses binding sites for substrate and allosteric effectors, and each of the smaller, beta subunits contains a binuclear iron complex. The iron complex interacts with a specific tyrosine residue to form a tyrosyl free radical which is essential for activity. Previous work has identified two genes in the yeast Saccharomyces cerevisiae, RNR1 and RNR3, that encode alpha subunits and one gene, RNR2, that encodes a beta subunit. Here we report the identification of a second gene from this yeast, RNR4, that encodes a protein with significant similarity to the beta-subunit proteins. The phenotype of rnr4 mutants is consistent with that expected for a defect in ribonucleotide reductase; rnr4 mutants are supersensitive to the ribonucleotide reductase inhibitor hydroxyurea and display an S-phase arrest at their restrictive temperature. rnr4 mutant extracts are deficient in ribonucleotide reductase activity, and this deficiency can be remedied by the addition of exogenous Rnr4p. As is the case for the other RNR genes, RNR4 is induced by agents that damage DNA. However, Rnr4p lacks a number of sequence elements thought to be essential for iron binding, and mutation of the critical tyrosine residue does not affect Rnr4p function. These results suggest that Rnr4p is catalytically inactive but, nonetheless, does play a role in the ribonucleotide reductase complex.