Rapid Freeze-quench Electron Paramagnetic Resonance Research Articles

Detection of a Kinetically Competent Compound-I Intermediate in the Vancomycin Biosynthetic Enzyme OxyB.

Cytochrome P450 enzymes are abundantly encoded in microbial genomes. Their reactions have two general outcomes, one involving oxygen insertion via a canonical "oxygen rebound" mechanism and a second that diverts from this pathway and leads to a wide array of products, notably intramolecular oxidative cross-links. The antibiotic of-last-resort, vancomycin, contains three such cross-links, which are crucial for biological activity and are installed by the P450 enzymes OxyB, OxyA, and OxyC. The mechanisms of these enzymes have remained elusive in part because of the difficulty in spectroscopically capturing transient intermediates. Using stopped-flow UV/visible absorption and rapid freeze-quench electron paramagnetic resonance spectroscopies, we show that OxyB generates the highly reactive compound-I intermediate, which can react with a model vancomycin peptide substrate in a kinetically competent fashion to generate product. Our results have implications for the mechanism of OxyB and are in line with the notion that oxygen rebound and oxidative cross-links share early steps in their catalytic cycles.

Kinetic analysis of amino acid radicals formed in H2O2-driven CuI LPMO reoxidation implicates dominant homolytic reactivity

Lytic polysaccharide monooxygenases (LPMOs) have been proposed to react with both [Formula: see text] and [Formula: see text] as cosubstrates. In this study, the [Formula: see text] reaction with reduced Hypocrea jecorina LPMO9A (CuI-HjLPMO9A) is demonstrated to be 1,000-fold faster than the [Formula: see text] reaction while producing the same oxidized oligosaccharide products. Analysis of the reactivity in the absence of polysaccharide substrate by stopped-flow absorption and rapid freeze-quench (RFQ) electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) yields two intermediates corresponding to neutral tyrosyl and tryptophanyl radicals that are formed along minor reaction pathways. The dominant reaction pathway is characterized by RFQ EPR and kinetic modeling to directly produce CuII-HjLPMO9A and indicates homolytic O-O cleavage. Both optical intermediates exhibit magnetic exchange coupling with the CuII sites reflecting facile electron transfer (ET) pathways, which may be protective against uncoupled turnover or provide an ET pathway to the active site with substrate bound. The reactivities of nonnative organic peroxide cosubstrates effectively exclude the possibility of a ping-pong mechanism.

Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.

Escherichia coli class Ia ribonucleotide reductase (RNR) is composed of two subunits that form an active α2β2 complex. The nucleoside diphosphate substrates (NDP) are reduced in α2, 35 Å from the essential diferric-tyrosyl radical (Y122•) cofactor in β2. The Y122•-mediated oxidation of C439 in α2 occurs by a pathway (Y122 ⇆ [W48] ⇆ Y356 in β2 to Y731 ⇆ Y730 ⇆ C439 in α2) across the α/β interface. The absence of an α2β2 structure precludes insight into the location of Y356 and Y731 at the subunit interface. The proximity in the primary sequence of the conserved E350 to Y356 in β2 suggested its importance in catalysis and/or conformational gating. To study its function, pH-rate profiles of wild-type β2/α2 and mutants in which 3,5-difluorotyrosine (F2Y) replaces residue 356, 731, or both are reported in the presence of E350 or E350X (X = A, D, or Q) mutants. With E350, activity is maintained at the pH extremes, suggesting that protonated and deprotonated states of F2Y356 and F2Y731 are active and that radical transport (RT) can occur across the interface by proton-coupled electron transfer at low pH or electron transfer at high pH. With E350X mutants, all RNRs were inactive, suggesting that E350 could be a proton acceptor during oxidation of the interface Ys. To determine if E350 plays a role in conformational gating, the strong oxidants, NO2Y122•-β2 and 2,3,5-F3Y122•-β2, were reacted with α2, CDP, and ATP in E350 and E350X backgrounds and the reactions were monitored for pathway radicals by rapid freeze-quench electron paramagnetic resonance spectroscopy. Pathway radicals are generated only when E350 is present, supporting its essential role in gating the conformational change(s) that initiates RT and masking its role as a proton acceptor.

Rapid Freeze-Quench EPR Spectroscopy: Improved Collection of Frozen Particles.

Rapid freeze-quench (RFQ) in combination with electron paramagnetic resonance (EPR) spectroscopy at X-band is a proven technique to trap and characterize paramagnetic intermediates of biochemical reactions. Preparation of suitable samples is still cumbersome, despite many attempts to remedy this problem, and limits the wide applicability of RFQ EPR. We present a method, which improves the collection of freeze-quench particles from isopentane and their packing in an EPR tube. The method is based on sucking the particle suspension into an EPR tube with a filter at the bottom. This procedure results in a significant reduction of the required volume of reactants, which allows the economical use of valuable reactants such as proteins. The approach also enables the successful collection of smaller frozen particles, which are generated at higher flow rates. The method provides for a reproducible, efficient and fast collection of the freeze-quench particles and can be easily adapted to RFQ EPR at higher microwave frequencies than X-band.

Reverse Electron Transfer Completes the Catalytic Cycle in a 2,3,5-Trifluorotyrosine-Substituted Ribonucleotide Reductase.

Escherichia coli class Ia ribonucleotide reductase is composed of two subunits (α and β), which form an α2β2 complex that catalyzes the conversion of nucleoside 5′-diphosphates to deoxynucleotides (dNDPs). β2 contains the essential tyrosyl radical (Y122•) that generates a thiyl radical (C439•) in α2 where dNDPs are made. This oxidation occurs over 35 Å through a pathway of amino acid radical intermediates (Y122 → [W48] → Y356 in β2 to Y731 → Y730 → C439 in α2). However, chemistry is preceded by a slow protein conformational change(s) that prevents observation of these intermediates. 2,3,5-Trifluorotyrosine site-specifically inserted at position 122 of β2 (F3Y•-β2) perturbs its conformation and the driving force for radical propagation, while maintaining catalytic activity (1.7 s–1). Rapid freeze–quench electron paramagnetic resonance spectroscopy and rapid chemical-quench analysis of the F3Y•-β2, α2, CDP, and ATP (effector) reaction show generation of 0.5 equiv of Y356• and 0.5 equiv of dCDP, both at 30 s–1. In the absence of an external reducing system, Y356• reduction occurs concomitant with F3Y reoxidation (0.4 s–1) and subsequent to oxidation of all α2s. In the presence of a reducing system, a burst of dCDP (0.4 equiv at 22 s–1) is observed prior to steady-state turnover (1.7 s–1). The [Y356•] does not change, consistent with rate-limiting F3Y reoxidation. The data support a mechanism where Y122• is reduced and reoxidized on each turnover and demonstrate for the first time the ability of a pathway radical in an active α2β2 complex to complete the catalytic cycle.

Equilibration of tyrosyl radicals (Y356•, Y731•, Y730•) in the radical propagation pathway of the Escherichia coli class Ia ribonucleotide reductase.

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric tyrosyl radical (Y(122)(•)) 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 (Y(122)(•) ⇆ [W(48)?] ⇆ Y(356) within β to Y(731) ⇆ Y(730) ⇆ C(439) within α). Previously, we reported that a β2 mutant with 3-nitrotyrosyl radical (NO(2)Y(•); 1.2 radicals/β2) in place of Y(122)(•) in the presence of α2, CDP, and ATP catalyzes formation of 0.6 equiv of dCDP and accumulates 0.6 equiv of a new Y(•) proposed to be located on Y(356) in β2. We now report three independent methods that establish that Y(356) is the predominant location (85-90%) of the radical, with the remaining 10-15% delocalized onto Y(731) and Y(730) 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%) and 33 ± 0.4 and 38 ± 0.5 Å (12% ± 3%) indicative of NO(2)Y(122)(•)-Y(356)(•), NO(2)Y(122)(•)-NO(2)Y(122)(•), and NO(2)Y(122)(•)-Y(731(730))(•), respectively. Radical distribution in α2 was supported by RFQ electron paramagnetic resonance (EPR) studies using Y(731)(3,5-F(2)Y) or Y(730)(3,5-F(2)Y)-α2, which revealed F(2)Y(•), studies using globally incorporated [β-(2)H(2)]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(•) radicals in the PCET pathway and their conformations.

Site(s) of Tyrosyl Radical Formation in Dehaloperoxidase A and B

Dehaloperoxidase (DHP) is a dual-function hemoprotein that, in addition to being the putative coelomic hemoglobin of Amphitrite ornata, exhibits a broad substrate specificity for the catalytic oxidation of mono-, di-, and trisubstituted halophenols, thus distinguishing DHP as the first globin shown to possess a biologically relevant peroxidase activity. Both isoenzymes of DHP, termed A and B, have been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that utilizes hydrogen peroxide as the oxidant. We have shown that the initially formed heme intermediate in wild-type DHP is not Compound I as is often the case in peroxidases, but rather is a combination of an iron(IV)-oxo and a tyrosyl radical that together have similarity to the Compound ES intermediate of cytochrome c peroxidase. In order to possibly identify the site(s) of this radical species in DHP, we have studied the tyrosine mutants DHP A (Y34F), DHP A (Y38F), DHP A (Y34F/Y38F), DHP B (Y28F), DHP B (Y38F), and DHP B (Y28F/Y38F), and studied their reaction with hydrogen peroxide using a combination of stopped-flow UV-visible and rapid-freeze quench electron paramagnetic resonance spectroscopies. Spectroscopic evidence for the formation of both Compound I and Compound ES will be presented. We have further characterized these mutants using biochemical assays to determine their effect on the catalytic activity of the enzyme, and relate these results to the structure of the heme active site and the formation of the catalytically-attenuated species Compound RH. Such mutagenesis studies of DHP provide critical insight into the mechanistic details of the H2O2-dependent oxidative dehalogenation reaction catalyzed by dehaloperoxidase, present a clearer description of the function of DHP at the molecular level, and lead to a better understanding of the paradigms of globin structure-function relationships.

Spectroscopic and Mechanistic Investigations of Dehaloperoxidase B fromAmphitrite ornata

Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. Of the two DHP isoenzymes identified to date, much of the recent focus has been on DHP A, whereas very little is known pertaining to the activity, substrate specificity, mechanism of function, or spectroscopic properties of DHP B. Herein, we report the recombinant expression and purification of DHP B, as well as the details of our investigations into its catalytic cycle using biochemical assays, stopped-flow UV-visible, resonance Raman, and rapid freeze-quench electron paramagnetic resonance spectroscopies, and spectroelectrochemistry. Our experimental design reveals mechanistic insights and kinetic descriptions of the dehaloperoxidase mechanism which have not been previously reported for isoenzyme A. Namely, we demonstrate a novel reaction pathway in which the products of the oxidative dehalogenation of trihalophenols (dihaloquinones) are themselves capable of inducing formation of oxyferrous DHP B, and an updated catalytic cycle for DHP is proposed. We further demonstrate that, unlike the traditional monofunctional peroxidases, the oxyferrous state in DHP is a peroxidase-competent starting species, which suggests that the ferric oxidation state may not be an obligatory starting point for the enzyme. The data presented herein provide a link between the peroxidase and oxygen transport activities which furthers our understanding of how this bifunctional enzyme is able to unite its two inherent functions in one system.

Characterization of the Radical Intermediates of Dehaloperoxidase A and B from Amphitrite Ornata

The enzyme intermediates of dehaloperoxidase (DHP) from the marine worm Amphitrite ornata are unique within both the globin and cytochrome c peroxidase superfamilies. Both isoenzymes of DHP, termed A and B, have been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that utilizes hydrogen peroxide as a co-substrate. We have shown that the initially formed heme intermediate in this reaction is not Compound I as is often the case in peroxidases, but rather is a combination of an iron(IV)-oxo (Compound II) and a tyrosyl radical that together have similarity to the Compound ES intermediate of cytochrome c peroxidase. In order to possibly identify the origin of this radical species in DHP, we have expressed the tyrosine mutants DHP A (Y34F), DHP A (Y38F), DHP A (Y34F/Y38F), and DHP B (Y38F), and studied their reaction with hydrogen peroxide using a combination of stopped-flow UV-visible and rapid-freeze quench electron paramagnetic resonance spectroscopies. Although each mutant exhibited an average signal at g ≈ 2.0058 confirming the presence of a protein radical, significant differences in the lineshape and width of each radical was observed. We have further characterized these mutants using biochemical assays to determine their effect on the catalytic activity of the enzyme, and relate these results to the structure of the heme active site. Such mutagenesis studies of DHP provide critical insight into the mechanistic details of the H2O2-dependent oxidative dehalogenation reaction catalyzed by dehaloperoxidase, present a clearer description of the function of DHP at the molecular level, and lead to a better understanding of the paradigms of globin structure-function relationships.

A Calibration Reaction for Rapid Freeze-Quench W-Band EPR

A simple setup for rapid freeze-quench electron paramagnetic resonance (EPR) at W-band is described. It is based on a BioLogic commercial apparatus and a modified sample collection appropriate for W-band capillaries. The standard reaction of myoglobin with azide, which converts high-spin Fe(III) to low-spin Fe(III), used for calibration of rapid freeze-quench X-band EPR is very inconvenient for high-field measurements. Here we propose a different simple calibration reaction for W-band: the reduction of a nitroxide free radical with sodium dithionite using Mn2+ as an internal standard. Using this calibration reaction we determined the dead time of our system to be less than 10 ms.

Radical Sites in Mycobacterium tuberculosis KatG Identified Using Electron Paramagnetic Resonance Spectroscopy, the Three-dimensional Crystal Structure, and Electron Transfer Couplings

Catalase-peroxidase (KatG) from Mycobacterium tuberculosis, a Class I peroxidase, exhibits high catalase activity and peroxidase activity with various substrates and is responsible for activation of the commonly used antitubercular drug, isoniazid (INH). KatG readily forms amino acid-based radicals during turnover with alkyl peroxides, and this work focuses on extending the identification and characterization of radicals forming on the millisecond to second time scale. Rapid freeze-quench electron paramagnetic resonance spectroscopy (RFQ-EPR) reveals a change in the structure of the initially formed radical in the presence of INH. Heme pocket binding of the drug and knowledge that KatG[Y229F] lacks this signal provides evidence for radical formation on residue Tyr(229). High field RFQ-EPR spectroscopy confirmed a tryptophanyl radical signal, and new analyses of X-band RFQ-EPR spectra also established its presence. High field EPR spectroscopy also confirmed that the majority radical species is a tyrosyl radical. Site-directed mutagenesis, along with simulations of EPR spectra based on x-ray structural data for particular tyrosine and tryptophan residues, enabled assignments based on predicted hyperfine coupling parameters. KatG mutants W107F, Y229F, and the double mutant W107F/Y229F showed alteration in type and yield of radical species. Results are consistent with formation of a tyrosyl radical reasonably assigned to residue Tyr(229) within the first few milliseconds of turnover. This is followed by a mixture of tyrosyl and tryptophanyl radical species and finally to only a tyrosyl radical on residue Tyr(353), which lies more distant from the heme. The radical processing of enzyme lacking the Trp(107)-Tyr(229)-Met(255) adduct (found as a unique structural feature of catalase-peroxidases) is suggested to be a reasonable assignment of the phenomena.

Rapid Formation of Compound II and a Tyrosyl Radical in the Y229F Mutant of Mycobacterium tuberculosis Catalase-peroxidase Disrupts Catalase but Not Peroxidase Function

Catalase-peroxidases (KatG), which belong to Class I heme peroxidase enzymes, have high catalase activity and substantial peroxidase activity. The Y229F mutant of Mycobacterium tuberculosis KatG was prepared and characterized to investigate the functional role of this conserved residue unique to KatG enzymes. Purified, overexpressed KatG[Y229F] exhibited severely reduced steady-state catalase activity while the peroxidase activity was enhanced. Optical stopped-flow experiments showed rapid formation of Compound (Cmpd) II (oxyferryl heme intermediate) in the reaction of resting KatG[Y229F] with peroxyacetic acid or chloroperoxybenzoic acid, without detectable accumulation of Cmpd I (oxyferryl heme pi-cation radical intermediate), the latter being readily observed in the wild-type enzyme under similar conditions. Facile formation of Cmpd III (oxyferrous enzyme) also occurred in the mutant in the presence of micromolar hydrogen peroxide. Thus, the lost catalase function may be explained in part because of formation of intermediates that do not participate in catalatic turnover. The source of the reducing equivalent required for generation of Cmpd II from Cmpd I was shown by rapid freeze-quench electron paramagnetic resonance spectroscopy to be a tyrosine residue, just as in wild-type KatG. The kinetic coupling of radical generation and Cmpd II formation was shown in KatG[Y229F]. Residue Y229, which is a component of a newly defined three amino acid adduct in catalase-peroxidases, is critically important for protecting the catalase activity of KatG.

Identification and Characterization of Tyrosyl Radical Formation in Mycobacterium tuberculosisCatalase-Peroxidase (KatG)

The catalytic function of Mycobacterium tuberculosis catalase-peroxidase (KatG) and its role in activation of the anti-tuberculosis antibiotic isoniazid were investigated using rapid freeze-quench electron paramagnetic resonance (RFQ-EPR) experiments. The reaction of KatG with peroxyacetic acid was followed as a function of time using x-band EPR at 77 K. A doublet EPR signal appears within 6.4 ms after mixing and at time points through hundreds of milliseconds. Thereafter, a singlet signal develops and finally predominates after 1 s, with a total yield of radical approximately 0.5 spin/heme. Simulation of the spectra provided EPR parameters consistent with those for tyrosyl radicals. Changes in the hyperfine splitting and/or line width in spectra for l-3,3-[2H2]tyrosine-labeled, but not l-2,4,5,6,7-[2H5]tryptophan-labeled KatG confirmed this assignment. The initial rate of radical formation was unchanged using a 3-fold or 10-fold excess of peroxyacetic acid, consistent with a rate-determining step involving an intermediate. Although Compound I is likely to be the precursor of tyrosyl radical in KatG, neither its EPR signal nor its reduction to Compound II during formation of the radical(s) could be observed. The tyrosyl radical doublet signal was rapidly quenched by addition of isoniazid and benzoic hydrazide, but not by iproniazid, which binds poorly to KatG.

An Improved Sample Packing Device for Rapid Freeze-Trap Electron Paramagnetic Resonance Spectroscopy Kinetic Measurements

A new method has been developed for sample packing in rapid freeze–quench electron paramagnetic resonance spectroscopy (EPR) kinetic experiments. Sample particles freeze–quenched in chilled isopentane are filtered under pressure through a stainless steel funnel attached to an EPR tube fitted with a porous disk at its bottom. Isopentane exits through the porous disk and the sample particles can be transferred essentially quantitatively into the receiving EPR tube. This device provides a more predictable, reproducible, and time-saving method for sample packing, enables use of a wider range of flow velocity, and allows efficient use of valuable reactants.

Kinetic Evidence That a Radical Transfer Pathway in Protein R2 of Mouse Ribonucleotide Reductase Is Involved in Generation of the Tyrosyl Free Radical

Class I ribonucleotide reductases consist of two subunits, R1 and R2. The active site is located in R1; active R2 contains a diferric center and a tyrosyl free radical (Tyr.), both essential for enzymatic activity. The proposed mechanism for the enzymatic reaction includes the transport of a reducing equivalent, i.e. electron or hydrogen radical, across a 35-A distance between Tyr. in R2 and the active site in R1, which are connected by a hydrogen-bonded chain of conserved, catalytically essential amino acid residues. Asp266 and Trp103 in mouse R2 are part of this radical transfer pathway. The diferric/Tyr. site in R2 is reconstituted spontaneously by mixing iron-free apoR2 with Fe(II) and O2. The reconstitution reaction requires the delivery of an external reducing equivalent to form the diferric/Tyr. site. Reconstitution kinetics were investigated in mouse apo-wild type R2 and the three mutants D266A, W103Y, and W103F by rapid freeze-quench electron paramagnetic resonance with >/=4 Fe(II)/R2 at various reaction temperatures. The kinetics of Tyr. formation in D266A and W103Y is on average 20 times slower than in wild type R2. More strikingly, Tyr. formation is completely suppressed in W103F. No change in the reconstitution kinetics was found starting from Fe(II)-preloaded proteins, which shows that the mutations do not affect the rate of iron binding. Our results are consistent with a reaction mechanism using Asp266 and Trp103 for delivery of the external reducing equivalent. Further, the results with W103F suggest that an intact hydrogen-bonded chain is crucial for the reaction, indicating that the external reducing equivalent is a H. Finally, the formation of Tyr. is not the slowest step of the reaction as it is in Escherichia coli R2, consistent with a stronger interaction between Tyr. and the iron center in mouse R2. A new electron paramagnetic resonance visible intermediate named mouse X, strikingly similar to species X found in E. coli R2, was detected only in small amounts under certain conditions. We propose that it may be an intermediate in a side reaction leading to a diferric center without forming the neighboring Tyr.

Mechanism of carbon monoxide oxidation by the carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum: kinetic characterization of the intermediates.

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Clostridium thermoaceticum catalyzes (i) the synthesis of acetyl-CoA from a methylated corrinoid protein, CO, and coenzyme A and (ii) the oxidation of CO to CO2. CO oxidation occurs at a Ni- and FeS-containing center known as cluster C. Electrons are transferred from cluster C to a separate metal center, cluster B, to external acceptors like ferredoxin. In the work described here, we performed reductive titrations of CODH/ACS with CO and sodium dithionite and monitored the reaction by electron paramagnetic resonance (EPR) spectroscopy. We also performed pre-steady-state kinetic studies by rapid freeze-quench EPR spectroscopy (FQ-EPR) and stopped-flow kinetics. Redox titrations of CODH/ACS revealed the existence of a UV-visible and EPR-silent electron acceptor denoted center S that does not appear to be associated with any of the other metal centers in the protein. Our results support the previous proposals [Anderson, M. E., & Lindahl, P. A. (1994) Biochemistry 33, 8702-8711; Anderson, M. E., & Lindahl, P. A. (1996) Biochemistry 35, 8371-8380] that the Cred2 form of cluster C is two electrons more reduced than the Cred1 form. The combined results from titrations and pre-steady-state studies were used to formulate a mechanism for CO oxidation, composed of the following steps: (i) CO binding to the [Cred1,Box, Xox] state to yield a Cred1-CO complex; (ii) two-electron reduction of Cred1 to Cred2 concerted with CO2 release; (iii) binding of a second CO molecule to the [Cred2,Box,Xox] state to form a Cred2-CO complex; (iv) electron transfer from Cred2-CO to cluster B to form [Cred2,Bred,Xred] with concerted release of the second CO2. Step iii competes with internal electron transfer from Cred2 to Box and Xox. At high CO concentrations, step iii is favored, whereas at low concentrations, only one CO molecule per turnover binds and undergoes oxidation. Closure of the catalytic cycle involves electron transfer from reduced enzyme to an electron acceptor protein, like ferredoxin. Xox is a yet-uncharacterized electron acceptor that may be an intermediate in the reduction of center S. The Cred2 state appears to be the predominant state of cluster C during steady-state turnover. The rate-determining step for the first half-reaction is step iv, while during steady-state turnover, it appears to be electron transfer to external electron acceptors.

Mechanism of assembly of the tyrosyl radical-dinuclear iron cluster cofactor of ribonucleotide reductase.

Incubation of the apoB2 subunit of Escherichia coli ribonucleotide reductase with Fe2+ and O2 produces native B2, which contains the tyrosyl radical-dinuclear iron cluster cofactor required for nucleotide reduction. The chemical mechanism of this reconstitution reaction was investigated by stopped-flow absorption spectroscopy and by rapid freeze-quench EPR (electron paramagnetic resonance) spectroscopy. Two novel intermediates have been detected in the reaction. The first exhibits a broad absorption band centered at 565 nanometers. Based on known model chemistry, this intermediate is proposed to be a mu-peroxodiferric complex. The second intermediate exhibits a broad absorption band centered at 360 nanometers and a sharp, isotropic EPR signal with g = 2.00. When the reaction is carried out with 57Fe2+, this EPR signal is broadened, demonstrating that the intermediate is an iron-coupled radical. Variation of the ratio of Fe2+ to B2 in the reaction and comparison of the rates of formation and decay of the intermediates to the rate of formation of the tyrosyl radical (.Y122) suggest that both intermediates can generate .Y122. This conclusion is supported by the fact that both intermediates exhibit an increased lifetime in a mutant B2 subunit (B2-Y122F) lacking the oxidizable Y122. Based on these kinetic and spectroscopic data, a mechanism for the reaction is proposed. Unlike reactions catalyzed by heme-iron peroxidases, oxygenases, and model complexes, the reconstitution reaction appears not to involve high-valent iron intermediates.