ENDOR spectroscopy and DFT calculations: evidence for the hydrogen-bond network within α2 in the PCET of E. coli ribonucleotide reductase.

Escherichia coli class I ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two subunits: α2 and ß2. ß2 contains a stable di-iron tyrosyl radical (Y(122)(•)) cofactor required to generate a thiyl radical (C(439)(•)) 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: Y(122) ⇆ W(48)[?] ⇆ Y(356) in ß2, across the subunit interface to Y(731) ⇆ Y(730) ⇆ C(439) in α2. Within α2 a colinear PCET model has been proposed. To obtain evidence for this model, 3-amino tyrosine (NH(2)Y) replaced Y(730) in α2, and this mutant was incubated with ß2, cytidine 5'-diphosphate, and adenosine 5'-triphosphate to generate a NH(2)Y(730)(•) in D(2)O. [(2)H]-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 C(439) and Y(731). 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 NH(2)Y(730)(•) are consistent with the combined effect of the hydrogen bonds to Cys(439) and Tyr(731), both nearly perpendicular to the ring plane of NH(2)Y(730.) 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 are available.

Structural examination of the transient 3-aminotyrosyl radical on the PCET pathway of E. coli ribonucleotide reductase by multifrequency EPR spectroscopy.

E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 A from a tyrosyl radical (Y(122)*) within the beta2 subunit to a cysteine residue (C(439)) within the alpha2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y(122) --> W(48) --> Y(356) in beta2, across the subunit interface to Y(731) --> Y(730) --> C(439) in alpha2. Using the suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodology, 3-aminotyrosine has been incorporated into position 730 in alpha2. Incubation of this mutant with beta2, substrate, and allosteric effector resulted in loss of the Y(122)* and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical (NH(2)Y*). In the current study [(15)N]- and [(14)N]-NH(2)Y(730)* have been generated in H(2)O and D(2)O 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 NH(2)Y(730)*. The g tensor (g(x) = 2.0052, g(y) = 2.0042, g(z) = 2.0022), the orientation of the beta-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 NH(2) moiety are consistent with an intramolecular hydrogen bond within NH(2)Y(730)*. This analysis is an essential first step in using the detailed structure of NH(2)Y(730)* to formulate a model for a PCET mechanism within alpha2 and for use of NH(2)Y in other systems where transient Y*s participate in catalysis.