E.coli class I ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two subunits: α2 and β2. β2 contains a stable di-iron tyrosyl radical (Y122•) cofactor required to generate a thiyl radical (C439•) in α2 over a distance of 35 Å, which in turn initiates the chemistry of the reduction process. The radical transfer process is proposed to occur by proton-coupled electron transfer (PCET) via a specific pathway: Y122 ⇆ W48[?] ⇆ Y356 in β2, across the subunit interface to Y731⇆ Y730 ⇆ C439 in α2. Within α2 a co-linear PCET model has been proposed. To obtain evidence for this model, 3-amino tyrosine (NH2Y) replaced Y730 in α2 and this mutant was incubated with β2, CDP and ATP to generate a (NH2Y730•) in D2O. [2H]-Electron-nuclear double resonance (ENDOR) spectra at 94 GHz of this intermediate were obtained and together with DFT models of α2 and quantum chemical calculations allowed assignment of the prominent ENDOR features to two hydrogen bonds likely associated with C439 and Y731. A third proton was assigned to a water molecule in close proximity (2.2 Å O-H---O distance) to residue 730. The calculations also suggest that the unusual g-values measured for NH2Y730• are consistent with the combined effect of the hydrogen bonds to Cys439 and Tyr731, both nearly perpendicular to the ring plane of NH2Y730. The results provide the first experimental evidence for the hydrogen bond network between the pathway residues in α2 of the active RNR complex, for which no structural data is available.

E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 Å from a tyrosyl radical (Y122•) within the β2 subunit to a cysteine residue (C439) within the α2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y122 → W48 → Y356 in β2, across the subunit interface to Y731 → Y730 → C439 in α2. Using the suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodology, 3-aminotyrosine has been incorporated into position 730 in α2. Incubation of this mutant with β2, substrate, and allosteric effector resulted in loss of the Y122• and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical (NH2Y•). In the current study [15N]- and [14N]-NH2Y730• have been generated in H2O and D2O and characterized by continuous wave 9 GHz EPR and pulsed EPR spectroscopies at 9, 94, and 180 GHz. The data give insight into the electronic and molecular structure of NH2Y730•. The g tensor (gx = 2.0052, gy = 2.0042, gz = 2.0022), the orientation of the β-protons, the hybridization of the amine nitrogen, and the orientation of the amino protons relative to the plane of the aromatic ring were determined. The hyperfine coupling constants and geometry of the NH2 moiety are consistent with an intramolecular hydrogen bond within NH2Y730•. This analysis is an essential first step in using the detailed structure of NH2Y730• to formulate a model for a PCET mechanism within α2 and for use of NH2Y in other systems where transient Y•s participate in catalysis.

For aromatic organic radicals, pulsed electron-electron double resonance (PELDOR) experiments at high magnetic fields provide information not only about the distance between the paramagnetic species but also about their relative orientation. However, the three-dimensional biradical structure is encoded in a complex pattern of orientation-selected PELDOR traces and the execution of the experiment is generally aggravated by constraints posed by the available hardware and the intrinsically low modulation depth observed. We present a 94 GHz PELDOR experiment performed with a commercial spectrometer and probe heads that permit separation of pump and detection frequencies up to 150 MHz. The setup is employed to examine the orientation selections on a general case of rigid biradicals with non-collinear g axes. The interacting radicals, a tyrosyl radical (Y 122 Á) located in the b2 subunit and an 3-aminotyrosyl radical (NH 2 Y 731 Á) located in the a2 subunit, are generated by Escherichia coli ribonucleotide reductase with a 3-aminotyrosine (NH 2 Y) site specifically incorporated into a2 in the presence of cytidine 5 0-diphosphate and adenosine 5 0-triphosphate. The experimental designs as well as some characteristic features of the observed modulation pattern are discussed.

First of all I would like to thank my supervisor Marina Bennati. I will always appreciate what she invested in me when I started to become part of one of the most interesting biochemical projects, the ribonucleotide reductase project. In EPR spectroscopy her view for details and her experience was vital for me and it was always impressing to see, to understand and to learn from her how far you need to go in order to come to the final conclusion. There are no parallels for her persistence in facing a scientific problem. I thank her in motivating me to go to MIT, in putting a lot of trust in me in order to face a challenging PhD project and in supporting me to push the high-field EPR/ENDOR investigations on E.coli RNR as far as possible.The research stay at MIT would not be feasible without JoAnne Stubbe. In her lab I was able to perform in a short time exciting science. Thank you a lot for this and for keeping our collaboration alive. Many, many thanks to Mohammad Seyedsayamdost, Clement Chan, Ellen Minnihan and Kenichi Yokoyama who gave many advices and who had always the right answer to my questions about RNR. Thanks also to Sumit Chakraborty, Rachael Buckley, Joey Cotruvo, Mimi Cho, Cha, Quamrul Hassan and many other former and recent members of the Stubbe group for spreading a good mood and giving me the temporary feeling to be part of.I am grateful to all my group members who had a big contribution to my work. First of all Giuseppe alias Emir Kusturica, I only can wish that you stay forever on the scientific road. Therefore let's use one of your famous sentences: "You can do everything, if you want!" I can only add to this: "You too, and long live Orazio!" Maria my PhD mate brought a bit of relaxation with her cool attitude into the group which sometimes could not be topped, even by our one and only Englishman and gentleman, Alistair. Thank you both for these rare moments."Igore, moj Igore!", thank you for tolerating my directness and sometimes unconventional style in saying my opinion. There were always delicate but also funny and honest moments between us.And Irina, thank you for keeping the group alive in your natural way and for being one of the big protagonists of the cocktail tour through Göttingen.A big "thank you" goes to Gitta, the heart of our group who has for each difficulty the right solution. Roberta I like the funny and interesting conversations during the Italian coffee breaks and Soraya I appreciate your cool and reserved nature.The newcomers Thomas, Nikolay, Roberto and Ines, I am sure you will make your way. I wish you good luck for your future projects and the will to succeed during your PhD. I am grateful also to my collaborators Prof. Frank Neese and Christoph Riplinger who made a huge effort in performing the DFT calculations on the proton-coupled electron transfer pathway of E.coli RNR. Especially Christoph I would like to thank for his never-ending patience and the spontaneous and numerous telephone calls, visits, SMS, Skype contacts which finally ended up in very nice scientific resu...