Equilibration of Tyrosyl Radicals (Y₃₅₆^•, Y₇₃₁^•, Y₇₃₀^•) in the Radical Propagation Pathway of the Escherichia coli Class Ia Ribonucleotide Reductase

Abstract

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric-tyrosyl radical (Y122•) cofactor in β2 to initiate catalysis in α2. Each turnover requires reversible long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y122• ⇆ [W48?] ⇆ Y356 within β to Y731 ⇆ Y730 ⇆ C439 within α). Previously, we reported that a β2 mutant with 3-nitrotyrosyl radical (NO2Y•, 1.2 radicals/β2) in place of Y122• in the presence of α2, CDP and ATP catalyzes formation of 0.6 equiv dCDP and accumulates 0.6 equiv of a new Y• proposed to be located on Y356 in β2. We now report three independent methods that establish that Y356 is the predominant location (85 – 90%) of the radical with the remaining 10 – 15% delocalized onto Y731 and Y730 in α2. Pulsed electron-electron double resonance spectroscopy on samples prepared by rapid freeze quench (RFQ) methods identified three distances: 30 ± 0.4 Å (88 ± 3%), 33 ± 0.4 Å and 38 ± 0.5 Å (12 ± 3%) indicative of NO2Y122•-Y356•, NO2Y122•- NO2Y122•, and NO2Y122•-Y731(730)•, respectively. Radical distribution in α2 was supported by RFQ EPR studies using Y731(3,5-F2Y) or Y730(3,5-F2Y)-α2 which revealed F2Y•, and by studies using globally incorporated [β-2H2]Y-α2 and analysis using parameters obtained from 140 GHz EPR spectroscopy. The amount of Y• delocalized in α2 from these two studies varied from 6 to 15%. The studies together give the first insight into the relative redox potentials of the three transient Y•s in the PCET pathway and their conformations.

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

“…; Table S5) (16)(17)(18)(19)(20)(21). A similar g-tensor anisotropy effect would be expected for a hydroxylated Trp radical because the radical spin density is typically delocalized over the entire indole ring (22)(23)(24)(25)(26)(27).…”

The prototypic class Ia ribonucleotide reductase from Escherichia coli: still surprising after all these years

“…This observation suggested the need for the radical to transfer through other residues to reach the surface of β 2 en route to the substrate-binding site of α 2 . Such a long-range electron transfer was unprecedented at the time it was proposed [8] but is now well supported by experimental evidence [19–21]. Another finding from the β 2 structure was that the 32 C-terminal residues were not observed due to their thermal lability (Fig.…”

“…Earlier studies demonstrated that C-terminal residues of β 2 were critical for its interaction with α 2 [22, 23]. Just upstream from this C-terminal sequence within the unstructured tail is the absolutely conserved Tyr356 that subsequent studies have shown to be part of the radical relay between the RNR subunits [19, 24, 25]. …”

“…More recently, incorporation of unnatural amino acids that act as radical traps at Tyr356, Tyr731, and Tyr730 has permitted the direct demonstration of redox-coupling between these residues [20, 21, 24]. Furthermore, these mutants enabled measurements of Tyr122-Tyr731 and Tyr122-Tyr730 distances by pulsed electron-electron double resonance (PELDOR) spectroscopy [19, 37]. These measured distances were consistent with those predicted by the α 2 β 2 docking model and thus provided the first experimental support for the subunit arrangement in this model.…”

Could tyrosine and tryptophan serve multiple roles in biological redox processes?

“…It is well established that multi-step tunnelling, called hopping, is required for functional charge transport in many redox enzymes (examples include ribonucleotide reductase [1][2][3][4][5][6][7][8][9], photosystem II [10][11][12], DNA photolyase [13][14][15][16][17][18][19][20][21], MauG [22][23][24][25] and cytochrome c peroxidase [26,27]). Here, we advance the hypothesis that many such enzymes, most especially those that generate high-potential intermediates during turnover, could be irreversibly damaged if the intermediates are not inactivated in some way.…”