High-throughput protein structural analysis using site-directed fluorescence labeling and the bimane derivative (2-pyridyl)dithiobimane

We present a site-directed fluorescence labeling (SDFL) study of 25 different T4 lysozyme protein samples labeled with the thiol-cleavable fluorophore, (2-pyridyl)dithiobimane (PDT-Bimane). Our results demonstrate PDT-Bimane can be used in cysteine-scanning studies to detect protein secondary structure, and to map proximity between sites in proteins by monitoring tryptophan quenching of bimane fluorescence. In addition, the reducible nature of PDT-Bimane can be exploited to resolve problems often faced in SDFL studies: ensuring specific labeling of cysteine residues, determining the extent of free label contamination, and accurately determining labeling efficiency even at low concentrations. The ability to cleave PDT-Bimane off the protein enables rapid determination of these parameters, and positions it as an ideal fluorophore for automated, high-throughput structural studies of protein folding, the detection of protein-protein interactions, and the monitoring of real-time conformational changes.     

Effect of ionic strength on the binding of ascorbate to albumin

A fluorophore-nitroxide free radical dual-functional probe (FN) was utilized to study the kinetics of ascorbate (AH(-)) binding to Bovine Serum Albumin (BSA). Since the free radical fragment in the FN probe intramolecularly quenches fluorescence, ascorbate reduction of the nitroxide function is accompanied by a concomitant fluorescence intensity increase from the fluorophore. Thus, both fluorescence and the EPR techniques could be utilized to measure the reaction rate. In the presence of BSA protein, the observed rate of the overall process is the sum of that from at least two reactions: the reaction between free ascorbate and free probe, and the reaction between bound ascorbate and bound probe. Our findings show that the observed rate is strongly dependent on the ionic strength of the medium. A corollary of this observation is the indication of a purely electrostatic interaction between ascorbate and the BSA protein. This conclusion was further corroborated by 1H NMR measurement of the transverse relaxation time, T(2), of ascorbate protons in BSA solutions. Ascorbate ion was released from the ascorbate/BSA ensemble in the presence of increasing concentrations of NaCl. Binding constants of AH(-) to BSA were calculated at different ionic strengths at pH 7.4. Furthermore, an increase in ionic strength did not affect the ability of albumin to protect ascorbate against autoxidation. This suggests that the protein's protective antioxidant effect may be attributed to BSA binding of trace quantities of transition-metal cations (rather than ascorbate binding to BSA). This conclusion is supported by ascorbate UV-absorption measurements in the presence of albumin and Cu(2+) ions as a function of ionic strength.     

Acrylodan-conjugated cysteine side chains reveal conformational state and ligand site locations of the acetylcholine-binding protein

We undertook cysteine substitution mutagenesis and fluorophore conjugation at selected residue positions to map sites of ligand binding and changes in solvent exposure of the acetylcholine-binding protein from Lymnaea stagnalis, a nicotinic receptor surrogate. Acrylodan fluorescence emission is highly sensitive to its local environment, and when bound to protein, exhibits changes in both intensity and emission wavelength that are reflected in the degree of solvent exclusion and the effective dielectric constant of the environment of the fluorophore. Hence, cysteine mutants were generated based on the acetylcholine-binding protein crystal structure and predicted ligand binding sites, and fluorescence parameters were assayed on the acrylodan-conjugated proteins. This approach allows one to analyze the environment around the conjugated fluorophore side chain and the changes induced by bound ligand. Introduction of an acrylodan-cysteine conjugate at position 178 yields a large blue shift with alpha-bungarotoxin association, whereas the agonists and alkaloid antagonists induce red shifts reflecting solvent exposure at this position. Such residue-selective changes in fluorescence parameters suggest that certain ligands can induce distinct conformational states of the binding protein, and that mutually exclusive binding results from disparate portals of entry to and orientations of the bound alpha-toxin and smaller acetylcholine congeners at the binding pocket. Labeling at other residue positions around the predicted binding pocket also reveals distinctive spectral changes for alpha-bungarotoxin, agonists, and alkaloid antagonists.     

Generation of Hydroxyl Radicals by Nucleohistone-Bound Metal-Adriamycin Complexes

A recently developed method has been utilized to demonstrate the generation of hydroxyl radicals (HO^bullet) in the immediate proximity of DNA by cop-per(II)/iron(HI)-adriamycin in the presence of ascorbate and hydrogen peroxide. SECCA, a succinylated derivative of coumarin, generates the fluorescent 7-hydroxy-SECCA following reaction with HO^bullet. SECCA was coupled to polylysine or to histone HI and then complexed to DNA. When HO' was generated in the proximity of DNA by polylysine-coupled iodine-125, which emits short range Auger electrons, 7-hydroxy-SECCA was produced. DMSO was only moderately efficient in reducing the fluorescence induction, demonstrating the “local” generation of HO^bullet in this system. Copper(II)/iron(III)-adriamycin in the presence of ascorbate and hydrogen peroxide generated the fluorescent 7-hydroxy-SECCA both when SECCA was free in solution and when SECCA was DNA-conjugated. With SECCA free in solution, the fluorescence induction was almost eliminated in the presence of HO^bullet scavengers (ethanol, tertbutanol or DMSO) and the relative efficiency of the scavengers in reducing the fluorescence followed their rate constant with HO^bullet. Furthermore, SECCA incubated with a singlet oxygen-generating compound demonstrated no fluorescence induction. When SECCA was positioned in close proximity to DNA as a SECCA-histone-H1-DNA complex, the relative efficiency of the scavengers in reducing the fluorescence still followed their rate constant with HO'; overall however the scavengers were much less effective in reducing the fluorescence, due presumably to the formation of HO^bullet radical in the immediate vicinity of DNA. These data suggest that copper(II)/iron(III)-adriamycin produces HO' in the presence of ascorbate and hydrogen peroxide whether unbound or bound to DNA and suggest that in the latter case scavengers would not prevent HO^bullet from attacking chromatin. In addition, the ability of DMSO to trap HO^bullet was shown to decrease as the conformation of the HI-DNA complex becomes more compact indicating the strong dependence of the trapping ability on chromatin conformation.     

Effect of albumin on the kinetics of ascorbate oxidation

The fluorescence intensity of the fluorophore in dansyl piperidine-nitroxide is intramolecularly quenched by the nitroxyl fragment. Therefore, the oxidation of ascorbic acid by the fluorophore-nitroxide (FN) probe can be monitored by two independent methods: steady-state fluorescence and electron paramagnetic resonance. Bovine serum albumin (BSA) affects the rate of this reaction. The influence of BSA on the rate is attributed to the adsorption of both ascorbate and the probe to BSA. Adsorption of ascorbate to BSA is confirmed by NMR relaxation experiments. The spatial distribution of the molecules on the BSA surface changes the availability of ascorbate and FN to each other. The results also point out that, in the presence of BSA, the autoxidation of ascorbate is significantly slowed down. The effect is studied at different pH values and explained in terms of the electrostatic interaction between the ascorbate anion and the BSA molecule.     

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels², ion pumps³, and transporters⁴. Recent developments have combined site-specific fluorophore labeling alongside TEVC to concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling^5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues^7,8. This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal⁵. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins⁴. Finally, recent developments have also enabled the manipulation of select internal ions following co-expression with a second protein⁹.Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (<5%)³ upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of the membrane protein in order to correlate the conformational dynamics to the function of the protein¹⁰. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.     

The reaction of ferric- and ferrous salts with bleomycin.

1. A comparative study shows that ferrous ions give a much better yield of Fe(III)-bleomycin than ferric ions, when iron salt is added to bleomycin in a buffer solution (pH 7.2). 2. The amount of Fe(III)-bleomycin formed after addition of ferric ions was markedly increased in the presence of ferric ion binding compounds (BSA, citrate) or reducing agents (ascorbate, cysteine).     

Conformational substates of myoglobin detected by extrinsic dynamic fluorescence studies

The extent of conformational substates of two apomyoglobins, i.e., sperm whale and tuna apomyoglobin, was investigated by examining the fluorescence decay in the frequency domain of the extrinsic fluorophore TNS [6-(p-toluidino)-2-naphthalenesulfonic acid] bound to the heme binding site. Data analysis was performed in terms of a continuous, unimodal lifetime distribution having a Lorentzian shape. The results were compared with those for the free fluorophore in an isotropic nonviscous solvent. The incorporation of TNS into the protein matrix resulted in a broadening of the lifetime distribution due to the microenvironmental heterogeneity generated by structural fluctuations. The larger width of lifetime distribution observed for TNS bound to tuna apomyoglobin was related to a more extended conformational space accessible to the fluorophore in this protein compared to sperm whale myoglobin. A temperature increase from 15 to 40 degrees C produced a further broadening of the lifetime distributions of TNS bound to both proteins. This result can be explained by assuming the existence of conformational substates at high energy content or separated by high energy barriers, which are not populated at low temperature. The overall picture emerging from the reported data is that the lifetime distributions of TNS bound to apomyoglobins are determined largely by the number of conformational substates accessible to the protein matrix and, to a lesser extent, by the interconversion rates among these states.     

A comparative study of adduct formation between the anticancer ruthenium(III) compound HInd trans-[RuCl4(Ind)2] and serum proteins

Formation of adducts between the antitumor ruthenium(III) complex [HInd]trans-[RuCl(4)(Ind)(2)] (KP1019) and the plasma proteins serum albumin and serum transferrin was investigated by UV-vis spectroscopy, for metal-to-protein ratios ranging from 1:1 to 5:1. In both cases, formation of tight metal-protein conjugates was observed. Similar spectroscopic features were observed for both albumin and transferrin derivatives implying a similar binding mode of the ruthenium species to these proteins. Surface histidines are the probable anchoring sites for the bound ruthenium(III) ions in line with previous crystallographic results. In order to assess the stability of the KP1019-protein adducts the influence of pH, reducing agents and chelators was analysed by UV-vis spectroscopy. Notably, there was no effect of addition of EDTA on the UV-vis spectra of the conjugates. The pH-stability was high in the pH range 5-8. Experiments with sodium ascorbate showed that there was just some alteration of selected bands. The implications of the present results are discussed in relation to the pharmacological behavior of this novel class of antitumor compounds.     

Dynamic fluorescence of extrinsic fluorophores as a tool for studying protein conformational substates

Summary The fluorescence lifetime distribution of 2-p-toluidinyl-6-naphthalene sulfonic acids (TNS) bound to the heme site of apomyoglobin has been examined. The results were compared to those observed for the free fluorophore in isotropic nonviscous solvent. Two different excitation wavelengths were used, i.e. 290 and 350 nm. The results showed that the distribution of TNS bound to apomyoglobin is wider than that of the free fluorophore, thus indicating the existence of a large number of conformational substates originating from the interaction between TNS and the protein matrix. The comparison of the distribution obtained at two different excitation wavelengths allowed the emission arising from conformational substates, in which the excited state of fluorophore moiety has a higher probability to be populated by Forster energy transfer mechanism, to be distinguished.     

Fluorescence quenching dynamics of tryptophan in proteins. Effect of internal rotation under potential barrier.

In many proteins fluorescence from single tryptophan exhibits a nonexponential decay function. To elucidate the origin of this nonexponential decay, we have examined the fluorescence decay function and time-resolved fluorescence anisotropy of a fluorophore covalently bound to a macromolecule by solving a rotational analogue of the Smoluchowski equation. An angular-dependent quenching constant and potential energy for the fluorophore undergoing internal rotation were introduced into the equation of motion for fluorophore. Results of numerical calculations using the equations thus obtained predict that both the fluorescence decay function and time-resolved anisotropy are dependent on rotational diffusion coefficients of fluorophore and potential energy for the internal rotation. The method was applied to the observed fluorescence decay curve of the single tryptophan in apocytochrome c from horse heart. The calculated decay curves fit the observed ones well.     

Solvent perturbation of the fluorescence of fluorescein bound to specific antibody. Fluorescence quenching of the bound fluorophore by iodide

Differential accessibility of liganded, high affinity rabbit anti-fluorescyl IgG antibody combining sites to the aqueous milieu has been investigated by solvent perturbation of the extrinsic fluorescence of bound fluorophore. Iodide, a dynamic quencher of fluorescein, was selected for use in these studies after examination of a number of water-soluble fluorescence quenchers. Quenching of antibody-bound fluorophore by iodide was measured with a number of liganded anti-fluorescyl IgG preparations, demonstrating partial solvent exposure of the fluorophore as well as heterogeneity of the high affinity antibody populations. Fluorescence quenching, lifetime, and absorption spectroscopy provided evidence that the antibody-bound fluorophore quenched by iodide interacted with it directly and that anomalous binding of the anion to the surface of the protein, resulting in ground state perturbations of the immunoglobulin, could not explain the observed results.     

Specific cross-linking of Lys233 and Cys235 in the mu opioid receptor by a reporter affinity label

The first example of the use of a reporter affinity label (NNA) that contains a fluorogenic naphthalene dialdehyde moiety to identify neighboring lysine and cysteine residues at a recognition site is described. The opioid receptors have served as the proof-of-concept because they contain multiple lysine and cysteine residues. The kinetics of isoindole formation resulting from covalent binding of NNA to wild-type and mutant opioid receptors were followed in cultured cells using flow cytometry. The finding that NNA bound to mutant mu opioid receptors (K233R and C235S) without producing specific fluorescence enhancement suggested that covalent bonding occurred at these positions to produce an isoindole fluorophore in the wild-type mu receptor. The similar kinetics of fluorophore formation for wild-type mu, delta, and kappa opioid receptors suggest that these conserved residues are the cross-linking sites in all three types of opioid receptors. The combined utilization of a reporter affinity label and site-directed mutagenesis offers a more expeditious method of identifying cross-linking at a recognition site when compared to classical procedures.     

Cell surface redox potential as a mechanism of defense against photosensitizers in fungi.

The phytotoxin cercosporin, a singlet oxygen-generating photosensitizer, is toxic to plants, mice, and many fungi, yet the fungi that produce it, Cercospora spp., are resistant. We hypothesize that resistance to cercosporin may result from a reducing environment at the cell surface. Twenty tetrazolium dyes differing in redox potential were used as indicators of cell surface redox potential of seven fungal species differing in resistance to cercosporin. Resistant fungi were able to reduce significantly more dyes than were sensitive fungi. A correlation between dye reduction and cercosporin resistance was also observed when resistance levels of Cercospora species were manipulated by growth on different media. The addition of the reducing agents ascorbate, cysteine, and reduced glutathione (GSH) to growth media decreased cercosporin toxicity for sensitive fungi. None of these agents directly reduced cercosporin at the concentrations at which they protected fungi. Spectral and thin-layer chromatographic analyses of cercosporin solutions containing the different reducing agents indicated that GSH, but not cysteine or ascorbate, reacted with cercosporin. Resistant and sensitive fungi did not differ in endogenous levels of cysteine, GSH, or total thiols. On the basis of data from this and other studies, this report presents a model which proposes that cercosporin resistance results from the production of reducing power at the surfaces of resistant cells, leading to transient reduction and detoxification of the cercosporin molecule.     

Cell surface redox potential as a mechanism of defense against photosensitizers in fungi.

The phytotoxin cercosporin, a singlet oxygen-generating photosensitizer, is toxic to plants, mice, and many fungi, yet the fungi that produce it, Cercospora spp., are resistant. We hypothesize that resistance to cercosporin may result from a reducing environment at the cell surface. Twenty tetrazolium dyes differing in redox potential were used as indicators of cell surface redox potential of seven fungal species differing in resistance to cercosporin. Resistant fungi were able to reduce significantly more dyes than were sensitive fungi. A correlation between dye reduction and cercosporin resistance was also observed when resistance levels of Cercospora species were manipulated by growth on different media. The addition of the reducing agents ascorbate, cysteine, and reduced glutathione (GSH) to growth media decreased cercosporin toxicity for sensitive fungi. None of these agents directly reduced cercosporin at the concentrations at which they protected fungi. Spectral and thin-layer chromatographic analyses of cercosporin solutions containing the different reducing agents indicated that GSH, but not cysteine or ascorbate, reacted with cercosporin. Resistant and sensitive fungi did not differ in endogenous levels of cysteine, GSH, or total thiols. On the basis of data from this and other studies, this report presents a model which proposes that cercosporin resistance results from the production of reducing power at the surfaces of resistant cells, leading to transient reduction and detoxification of the cercosporin molecule.     

Antibody-hapten interactions: circular and linear polarization of the fluorescence of dansyl bound to anti-dansyl antibodies

The fluorescence spectra of several dansyl derivatives (dansylamide, ?-N-dansyl-l-lysine, dansyl-l-alanine, and α-N-dansyl-l-alanine amide) bound to anti-dansyl antibodics (induced by an α-N-dansyl-poly d,l-alanine-poly l-lysine conjugate) are shifted by about 60 nm to the blue, and the quantum yields are markedly enhanced, compared to their respective fluorescence properties in water. The light emitted by the bound haptens is partly circularly polarized, reflecting the asymmetry induced in the bound chromophores by the antibody combining site. In contradistinction, the fluorescence spectrum of 1-dansyl-2-alanine diaminoethane bound to anti-alanine antibodies is similar to that of the free fluorophore in water and lacks circular polarization. These results imply that in this case the fluorophore of the hapten protrudes out of the site into the aqueous solvent. No circular dichroism is observed in the 300 to 400 nm region for the dansyl-anti-dansyl complex. Thus a change in the mode of interaction between the chromophore and its binding site takes place upon electronic excitation. The heterogeneity of the antibody binding sites is expressed by the dependence of the circular polarization of fluorescence on excitation wavelength. Differences in the circular polarization of luminescence were also observed when the residues attached to the dansyl group have been varied. This may reflect differences in the alignment of the fluorophore within the binding sites for the different dansyl derivatives.The linear polarization of dansylamide dissolved in glycerol is not constant across the emission band, indicating that the transition dipole moments related to the various vibronic states do not have the same spatial directions. Vibronic mixing of the emitting excited state with higher electronic states is thus indicated. Dansyl-l-alanine bound to anti-dansyl antibodies exhibitsan even more pronounced variation of the linear polarization across the emission band. In this case, the dependence of the linear polarization of the emitted light on excitation wavelength is anomalous, which is again a reflection of the heterogeneity of the population of the antibody molecules. The implications of these results to the studies of the fluorescence polarization of dansyl-protein complexes are discussed.     

Exploring membrane organization and dynamics by the wavelength-selective fluorescence approach

Wavelength-selective fluorescence comprises a set of approaches based on the red edge effect in fluorescence spectroscopy which can be used to directly monitor the environment and dynamics around a fluorophore in a complex biological system. A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of absorption band, is termed red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases where the dipolar relaxation time for the solvent shell around a fluorophore is comparable to or longer than its fluorescence lifetime. REES arises from slow rates of solvent relaxation (reorientation) around an excited state fluorophore which is a function of the motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. Utilizing this approach, it becomes possible to probe the mobility parameters of the environment itself (which is represented by the relaxing solvent molecules) using the fluorophore merely as a reporter group. Further, since the ubiquitous solvent for biological systems is water, the information obtained in such cases will come from the otherwise 'optically silent' water molecules. This makes REES and related techniques extremely useful since hydration plays a crucial modulatory role in a large number of important cellular events, including lipid-protein interactions and ion transport. The interfacial region in membranes, characterized by unique motional and dielectric characteristics, represents an appropriate environment for displaying wavelength-selective fluorescence effects. The application of REES and related techniques (wavelength-selective fluorescence approach) as a powerful tool to monitor the organization and dynamics of probes and peptides bound to membranes, micelles, and reverse micelles is discussed.     

Quenching of the intrinsic tryptophan fluorescence of mitochondrial ubiquinol--cytochrome-c reductase by the binding of ubiquinone

1. The quenching by ubiquinone (Q) of the intrinsic fluorescence of tryptophan residues within ubiquinol--cytochrome-c reductase (complex III) has been exploited to provide direct information on the interaction between these two components of the mitochondrial respiratory chain. 2. The fluorescence quenching data have been corrected for inner filter effects and interpreted using the classical Stern-Volmer and modified Stern-Volmer plots. The latter of these plots allows computation of both the dissociation constant (Kd) of complex formation between ubiquinone and complex III, and the percentage of fluorophores accessible to quenching. 3. It is found that different Q homologues bind to complex III with different affinities depending upon the length of the isoprenoid chain: 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone, an analogue of Q2, exhibits the same Kd as Q2. Furthermore, the accessibility of fluorophores to quenching was lower for Q1 than for the other quinones tested. 4. The binding affinity of Q2 to complex III depends upon the redox state of the enzyme. 5. Addition of the complex III inhibitor, antimycin, has very little effect on the binding affinity or on the accessibility of fluorophores to the quencher. 6. Addition of the inhibitor myxothiazol has a similar effect to reducing complex III with ascorbate. 7. Reconstitution of complex III into asolectin lipid vesicles gives similar qualitative results to the enzyme in solution regarding both the redox state and the addition of inhibitors.     

Formation of bile pigments by coupled oxidation of cobalt-substituted haemoglobin and myoglobin.

Treatment of cobalt-substituted haemoglobin and myoglobin with ascorbate and molecular O2 (coupled oxidation) resulted in biliverdin formation from the cobalt(II) derivatives but not from the cobalt(III) derivatives. This was apparently due to the inability of ascorbate to reduce cobalt(III) haemoproteins. Isomer analysis of the biliverdins produced from coupled oxidation of cobalt(II) oxyhaemoglobin suggested that the orientation of the cobalt protoporphyrin IX in the haem pocket differed slightly from that of the haem in native haemoglobin.     

Mechanistic studies of 1-aminocyclopropane-1-carboxylic acid oxidase: single turnover reaction

The final step in the biosynthesis of the plant hormone ethylene is catalyzed by the non-heme iron-containing enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). ACC is oxidized at the expense of O(2) to yield ethylene, HCN, CO(2), and two waters. Continuous turnover of ACCO requires the presence of ascorbate and HCO(3)(-) (or an alternative form), but the roles played by these reagents, the order of substrate addition, and the mechanism of oxygen activation are controversial. Here these issues are addressed by development of the first functional single turnover system for ACCO. It is shown that 0.35 mol ethylene/mol Fe(II)ACCO is produced when the enzyme is combined with ACC and O(2) in the presence of HCO(3)(-) but in the absence of ascorbate. Thus, ascorbate is not required for O(2) activation or product formation. Little product is observed in the absence of HCO(3)(-), demonstrating the essential role of this reagent. By monitoring the EPR spectrum of the sample during single turnover, it is shown that the active site Fe(II) oxidizes to Fe(III) during the single turnover. This suggests that the electrons needed for catalysis can be derived from a fraction of the initial Fe(II)ACCO instead of ascorbate. Addition of ascorbate at 10% of its K(m) value significantly accelerates both iron oxidation and ethylene formation, suggesting a novel high-affinity effector role for this reagent. This role can be partially mimicked by a non-redox-active ascorbate analog. A mechanism is proposed that begins with ACC and O(2) binding, iron oxidation, and one-electron reduction to form a peroxy intermediate. Breakdown of this intermediate, perhaps by HCO(3)(-)-mediated proton transfer, is proposed to yield a high-valent iron species, which is the true oxidizing reagent for the bound ACC.