A New Mechanism-based Radical Intermediate in a Mutant R1 Protein Affecting the Catalytically Essential Glu441 inEscherichia coli Ribonucleotide Reductase

Abstract

The invariant active site residue Glu 441 in protein R1 of ribonucleotide reductase from Escherichia coli has been engineered to alanine, aspartic acid, and glutamic acid. Each mutant protein was structurally and enzymatically characterized. Glu 441 contributes to substrate binding, and a carboxylate side chain at position 441 is essential for catalysis. The most intriguing results are the suicidal mechanism-based reaction intermediates observed when R1 E441Q is incubated with protein R2 and natural substrates (CDP and GDP). In a consecutive reaction sequence, we observe at least three clearly discernible steps: (i) a rapid decay (k 1 > 1. Ribonucleotide reductase is an essential enzyme of all living cells and catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides. Several classes of ribonucleotide reductases with different subunit composition and cofactor requirements are known, but they all share a radical-based reaction mechanism (1).The aerobic class Ia ribonucleotide reductase from Escherichia coli is the best characterized enzyme. It consists of two components denoted protein R1 and protein R2, each of which is a homodimer. Protein R1 contains redox-active cysteines essential for catalysis. Cysteines 225, 439, and 462 are located at the active site, where all four physiological substrates (CDP, UDP, GDP, or ADP) can bind. R1 also contains two different allosteric sites that bind nucleoside triphosphate effector molecules. One site regulates the overall enzyme activity, and the other site determines the substrate specificity (2, 3). Protein R2 contains a stable tyrosyl free radical at position 122 and an adjacent dinuclear iron center (4 -6). The tyrosyl radical is essential for catalysis.The separate three-dimensional structures of protein R1 and of protein R2 are known (6 -9). A model-built holoenzyme complex of the R1 and R2 structures indicates that the distance between the active site in R1 and Tyr 122 in R2 is about 30 -40 Å (8). Chains of conserved hydrogen-bonded residues leading from the active site of R1 in the direction of Tyr 122 in R2, and vice versa, have been identified and are believed to be part of a radical transfer pathway between the two sites (1, 6 -9). Mutational analysis of the residues postulated to be involved in radical transfer between R1 and R2 during catalysis supports this hypothesis (4, 10 -14).

Cysteines Involved in Radical Generation and Catalysis of Class III Anaerobic Ribonucleotide Reductase

“…Interestingly, Cys-290 of T4 NrdD and Cys-439 of R1 could also be aligned to Cys-419 of PFL, one of the cysteines shown to form a transient thiyl radical in the PFL system (14 -16). Recent results for the class I RNR indicate involvement of a conserved carboxylate side chain in the steps following the abstraction of the 3Ј-hydrogen of the substrate (37)(38)(39). No direct counterpart of this residue is found in the class III RNR, instead formate may take this role.…”

Spectroscopic and theoretical approaches for studying radical reactions in class I ribonucleotide reductase

“…In addition, a-eliminations of ketyl radical anions are rapid and efficient processes widely applied in synthesis (Schmittel and Ghorai, 2001). However, while solution studies clearly favor the radical anion mechanism due to its lower barrier than the radical cation variant (Lenz and Giese, 1997), results from mutants of E. coli argue strongly against a stepwise radical anion mechanism (Persson et al, 1997). In an E441Q R1 mutant study, Stubbe and colleagues shut down the deprotonation pathway for the 39-ribonucleotide radical by exchanging E441 (glutamate) with Q441 (glutamine), but formation of the disulfide radical anion was observed nevertheless (Lawrence et al, 1999).…”

“…The concept was first introduced by Zipse (1995), who suggested that a protonated glutamate could act as a bifunctional catalyst. A variation of this theme using only neutral molecules to avoid charge separation was computationally investigated slightly later (Siegbahn, 1998), but the experimental pH dependence of the enzyme activity (Persson et al, 1997), showing highest activity at pH 8, is more suggestive of the involvement of E441 in its deprotonated form wpK a(Glu-COOH) s4.4x. At about the same time, Zipse's hypothesis was refined by Stubbe and van der Donk (1998), who postulated that the E441 carboxylate group abstracts the proton at 39-OH simultaneously with the protonation of 29-OH by C225, leading to facile dehydration.…”

“…While the essential residues involved in the catalytic cycle have been identified through activity studies of sitedirected mutants (Mao et al, 1992a,b,c;Lawrence et al, 1999), the proximity of the most relevant residues, Cys 225 , Cys 439 , Cys 462 , Glu 441 , and Asn 437 , to the substrate C29 and C39 centers has been revealed through X-ray crystal structure analysis of the R1 protein with bound substrate (Uhlin and Eklund, 1994;Eriksson et al, 1997;Persson et al, 1997). These pieces of evidence were splendidly merged with results from experiments on small model systems in homogeneous solution, indicating that the elimination of water can indeed be effected via a radical pathway.…”

Biomimetic Modeling of the Nitrogen-centered Radical Postulated to occur during the Inhibition of Ribonucleotide Reductases by 2'-Azido-2'-deoxynucleotides.

“…With glu441 accepting a proton from the hydrogen of the hydroxyl group on C3' and cys225 donating a proton to the oxygen atom on C2', the radical on C3' is then transferred to C2' eliminating the hydroxyl group on C2' while releasing H 2 O and oxidizing the hydroxyl group on C3' to form 3'-keto-2-deoxyribonucleotides 3. 3,7,[21][22][23] The next step of the mechanism involves the radical on C2' abstracting the hydrogen atom on cys462, and cys225 donating the hydride ion to cys462 forming a disulfide radical anion 4. The radical from the cys225-cys462 is then transferred to C3' reducing the keto to hydroxyl with the help of glu441.…”