Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 ofE. coliRibonucleotide Reductase

Abstract

The Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric-tyrosyl radical cofactor for catalysis. RNR is composed of a 1:1 complex of two homodimeric subunits: α and β. Incubation of the E441Q-α mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale. The first radical was previously established by stopped flow UV/vis spectroscopy and pulsed high field EPR spectroscopy to be a disulfide radical anion. The second radical was proposed to be a 4′-radical of a 3′-keto-2′-deoxycytidine 5′-diphosphate. To identify the structure of the nucleotide radical [1′-2H], [2′-2H], [4′-2H], [5′-2H], [U−13C, 15N], [U−15N], and [5,6 -2H] CDP and [β-2H] cysteine-α were synthesized and incubated with E441Q-α2β2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35 GHz ENDOR spectroscopy. Substitution of 2H at C4′ and C1′ altered the observed hyperfine interactions of the nucleotide radical and established that the observed structure was not that predicted. DFT calculations (B3LYP/IGLO-III/B3LYP/TZVP) were carried out in an effort to recapitulate the spectroscopic observations and lead to a new structure consistent with all of the experimental data. The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5′-diphosphate. The relationship of this radical to the disulfide radical anion is discussed.

Nitrite and Hydroxylamine as Nitrogenase Substrates: Mechanistic Implications for the Pathway of N2 Reduction

“…Q-band ENDOR and ESEEM spectra were recorded at 2 K on CW 19 and pulsed 20,21 spectrometers. In both cases, the Q-band spectrum appears as the absorption envelope, rather than its derivative as in conventional EPR.…”

Unification of reaction pathway and kinetic scheme for N 2 reduction catalyzed by nitrogenase

“…Sample preparation followed freeze-quench procedures previously described (22,24,25); the 35 GHz CW (26) and pulsed (27) EPR spectrometers also have been described. Fourier transforms were performed with Bruker software, Win-EPR.…”

Composition and Structure of the Inorganic Core of Relaxed Intermediate X(Y122F) of Escherichia coli Ribonucleotide Reductase

“…26 As discussed in detail, 27–29 for a frozen solution sample, the determination of the full hyperfine tensor (and quadrupole tensor) of an interacting nucleus is achieved by obtaining a 2-D set of orientation-selective ENDOR spectra collected at multiple fields across the EPR envelope and comparing this set with simulated 2-D patterns. The ENDOR simulations were performed with the program Endorsim an enhanced version of the simulation program GENDOR.…”