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G Bar

Publications and source records attributed to G Bar.

6 recordsLinked to original sources
PubMed2001

Why multiple small subunits (Y2 and Y4) for yeast ribonucleotide reductase? Toward understanding the role of Y4.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides. Class I RNRs are composed of two homodimeric subunits: R1 and R2. R1 is directly involved in the reduction, and R2 contains the diferric-tyrosyl radical (Y*) cofactor essential for the initiation of reduction. Saccharomyces cerevisiae has two RNRs; Y1 and Y3 correspond to R1, whereas Y2 and Y4 correspond to R2. Y4 is essential for diferric-Y* formation in Y2 from apoY2, Fe(2+), and O(2). The actual function of Y4 is controversial. Y2 and Y4 have been further characterized in an effort to understand their respective roles in nucleotide reduction. (His)(6)-Y2, Y4, and (His)(6)-Y4 are homodimers, isolated largely in apo form. Their CD spectra reveal that they are predominantly helical. The concentrations of Y2 and Y4 in vivo are 0.5-2.3 microM, as determined by Western analysis. Incubation of Y2 and Y4 under physiological conditions generates apo Y2Y4 heterodimer, which can form a diferric-Y small middle dot when incubated with Fe(2+) and O(2). Holo Y2Y4 heterodimer contains 0.6-0.8 Y* and has a specific activity of 0.8-1.3 micromol.min.mg. Titration of Y2 with Y4 in the presence of Fe(2+) and O(2) gives maximal activity with one equivalent of Y4 per Y2. Models for the function of Y4 based on these data and the accompanying structure will be discussed.

Apoenzymes↗
PubMed2001

High-frequency (140-GHz) time domain EPR and ENDOR spectroscopy: the tyrosyl radical-diiron cofactor in ribonucleotide reductase from yeast.

High-frequency pulsed EPR and ENDOR have been employed to characterize the tyrosyl radical (Y*)-diiron cofactor in the Y2-containing R2 subunit of ribonucleotide reductase (RNR) from yeast. The present work represents the first use of 140-GHz time domain EPR and ENDOR to examine this system and demonstrates the capabilities of the method to elucidate the electronic structure and the chemical environment of protein radicals. Low-temperature spin-echo-detected EPR spectra of yeast Y* reveal an EPR line shape typical of a tyrosyl radical; however, when compared with the EPR spectra of Y* from E. coli RNR, a substantial upfield shift of the g(1)-value is observed. The origin of the shift in g(1) was investigated by 140-GHz (1)H and (2)H pulsed ENDOR experiments of the Y2-containing subunit in protonated and D(2)O-exchanged buffer. (2)H ENDOR spectra and simulations provide unambiguous evidence for one strongly coupled (2)H arising from a bond between the radical and an exchangeable proton of an adjacent residue or a water molecule. Orientation-selective 140-GHz ENDOR spectra indicate the direction of the hydrogen bond with respect to the molecular symmetry axes and the bond length (1.81 A). Finally, we have performed saturation recovery experiments and observed enhanced spin lattice relaxation rates of the Y* above 10 K. At temperatures higher than 20 K, the relaxation rates are isotropic across the EPR line, a phenomenon that we attribute to isotropic exchange interaction between Y* and the first excited paramagnetic state of the diiron cluster adjacent to it. From the activation energy of the rates, we determine the exchange interaction between the two irons of the cluster, J(exc) = -85 cm(-)(1). The relaxation mechanism and the presence of the hydrogen bond are discussed in terms of the differences in the structure of the Y*-diiron cofactor in yeast Y2 and other class I R2s.

Deuterium↗
PubMed2000

Two-dimensional ENDOR-ESEEM correlation spectroscopy

A two-dimensional (2D) experiment that correlates electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) frequencies, useful for unraveling and assigning ENDOR and ESEEM spectra from different paramagnetic centers with overlapping EPR spectra, is presented. The pulse sequence employed is similar to the Davies ENDOR experiment with the exception that the two-pulse echo detection is replaced by a stimulated echo detection in order to enhance the resolution in the ESEEM dimension. The two-dimensional data set is acquired by measuring the ENDOR spectrum as a function of the time interval T between the last two microwave pulses of the stimulated echo detection scheme. This produces a series of ENDOR spectra with amplitudes that are modulated with T. Fourier transformation (FT) with respect to T then generates a 2D spectrum with cross peaks connecting spectral lines of the ESEEM and ENDOR spectra that belong to the same paramagnetic center. Projections along the vertical and horizontal axes give the three-pulse FT-ESEEM and ENDOR spectra, respectively. The feasibility of the experiment was tested by simulating 2D ENDOR-ESEEM correlation spectra of a system consisting of an electron spin (S = (1/2)) coupled to two nuclei (I(1) = I(2) = (1/2)), taking into account experimental conditions such as pulse durations and off-resonance irradiation frequencies. The experiment is demonstrated on a single crystal of Cu(2+) doped l-histidine (Cu-His), containing two symmetrically related Cu(2+) sites that at an arbitrary orientation exhibit overlapping ESEEM and ENDOR spectra. While the ESEEM spectrum is relatively simple and arises primarily from one weakly coupled (14)N, the ENDOR spectrum is very crowded due to contributions from two nonequivalent nitrogens, two chlorides, and a relatively large number of protons. The simple ESEEM projection of the 2D ENDOR-ESEEM correlation spectrum is then used to disentangle the ENDOR spectrum and resolve two sets of lines corresponding to the different sites. Copyright 2000 Academic Press.

Journal Article↗
PubMed1999

High-field EPR detection of a disulfide radical anion in the reduction of cytidine 5'-diphosphate by the E441Q R1 mutant of Escherichia coli ribonucleotide reductase.

Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 and R2. The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y.) cofactor and R1 is the site of the conversion of nucleoside diphosphates to 2'-deoxynucleoside diphosphates. A mutant in the R1 subunit of Escherichia coli RNR, E441Q, was generated in an effort to define the function of E441 in the nucleotide-reduction process. Cytidine 5'-diphosphate was incubated with E441Q RNR, and the reaction was monitored by using stopped-flow UV-vis spectroscopy and high-frequency (140 GHz) time-domain EPR spectroscopy. These studies revealed loss of the Y. and formation of a disulfide radical anion and present experimental mechanistic insight into the reductive half-reaction catalyzed by RNR. These results support the proposal that the protonated E441 is required for reduction of a 3'-ketodeoxynucleotide by a disulfide radical anion. On the minute time scale, a second radical species was also detected by high-frequency EPR. Its g values suggest that this species may be a 4'-ketyl radical and is not on the normal reduction pathway. These experiments demonstrate that high-field time-domain EPR spectroscopy is a powerful new tool for deconvolution of a mixture of radical species.

Amino Acid Substitution↗
PubMed1999

Combined ultramicrotomy for AFM and TEM using a novel sample holder.

A new sample holder that allows combined microtomy for atomic force microscopy (AFM) and transmission electron microscopy (TEM) is described. The main feature of this sample holder is a small central part holding the sample. This central part fits into the head of an atomic force microscope. AFM measurements can be performed with a sample mounted in this central part of the sample holder. This makes the alignment of a microtomed bulk sample unnecessary, and offers the opportunity of an easy and fast combined sample preparation for AFM and TEM.

Microscopy, Atomic Force↗