Nature of the Peroxo Intermediate of the W48F/D84E Ribonucleotide Reductase Variant:  Implications for O₂ Activation by Binuclear Non-Heme Iron Enzymes

Abstract

Analysis of the spectroscopic signatures of the R2-W48F/D84E biferric peroxo intermediate identifies a cis mu-1,2 peroxo coordination geometry. DFT geometry optimizations on both R2-W48F/D84E and R2-wild-type peroxo intermediate models including constraints imposed by the protein also identify the cis mu-1,2 peroxo geometry as the most stable peroxo intermediate structure. This study provides significant insight into the electronic structure and reactivity of the R2-W48F/D84E peroxo intermediate, structurally related cis mu-1,2 peroxo model complexes, and other enzymatic biferric peroxo intermediates.

Human deoxyhypusine hydroxylase, an enzyme involved in regulating cell growth, activates O 2 with a nonheme diiron center

“…This mechanical coupling model is supported by a recent study of a series of synthetic (cis--1,2-peroxo)diiron(III) complexes (32). Indeed, a detailed spectroscopic and DFT analysis of the W48F/D84E R2 peroxo intermediate also questions the 2.5-Å FeϪFe distance deduced by EXAFS and favors an FeϪFe distance of 3.68 Å (18) that is closer to the 3.44 Å distance we have determined for hDOHH peroxo by EXAFS analysis. On the basis of the calculations, it is proposed that the R2 peroxo intermediate has a (cis--1,2-peroxo)diiron(III) center supported by 2 bidentate carboxylate bridges.…”

“…Such very short metalϪmetal separations are found only for synthetic complexes that have 2 or 3 single-atom bridges (42,43). Despite this apparent difference, these 3 diiron(III)-peroxo intermediates exhibit (O-O) Raman features of comparable frequency, at 855, 868 (observed for the peroxo intermediate of W48F/D84E R2 and assumed to resemble that of W48A/D84E R2 in structure), and 851 cm Ϫ1 , respectively (18,21). The similarity of the (O-O) values suggests that the diiron-peroxo core structures may not be as different as suggested by the EXAFS results.…”

“…From their studies of W48F/D84E R2, Skulan et al conclude that a cis--1,2-peroxo form is the most stable structure for the diiron(III)-peroxo intermediate and must undergo a structural change before O-O bond cleavage to generate the high valent iron species required for substrate oxidation (18). The additional single-atom bridge deduced to be present in the diiron(III)-peroxo unit in hDOHH peroxo (Fig.…”

“…Its blue chromophore persists for at least several days at room temperature, so its stability surpasses even that of peroxo ⌬ 9 D, the peroxo intermediate obtained from chemically reduced ⌬ 9 D, which has a half-life of Ϸ30 min at room temperature (14). Thus the lifetimes of diiron-peroxo intermediates of these enzymes vary significantly and can range from milliseconds to days (14)(15)(16)(17)(18)(19)(20)23). Interestingly, the decay of hDOHH peroxo is accelerated by the addition of its substrate, the deoxyhypusine-containing eIF5A.…”

“…This chromophore gives rise to 2 absorption features at 320 nm and 630 nm with molar extinction coefficients indicative of ligand-to-metal charge transfer transitions (8). These features resemble those associated with diiron(III)-peroxo intermediates of the hydroxylase component of methane monooxygenase (MMOH) (12), stearoyl-acyl carrier protein ⌬ 9 desaturase (⌬ 9 D) (13, 14), the R2 proteins of class I ribonucleotide reductases (R2) (15)(16)(17)(18)(19), and the ferroxidase site of frog M ferritin (20,21). Some of these intermediates have been characterized by resonance Raman spectroscopy and found to exhibit vibrational features typical of a ( -1,2-peroxo)diiron(III) unit (13,17,20).…”