Electron Paramagnetic Resonance Titration Research Articles

Characterization of the Membrane-Associated Electron-Bifurcating Flavoenzyme EtfABCX from the Hyperthermophilic Bacterium Thermotoga maritima.

Electron bifurcation is an energy-conservation mechanism in which a single enzyme couples an exergonic reaction with an endergonic one. Heterotetrameric EtfABCX drives the reduction of low-potential ferredoxin (E°' ∼ -450 mV) by oxidation of the midpotential NADH (E°' = -320 mV) by simultaneously coupling the reaction to reduction of the high-potential menaquinone (E°' = -74 mV). Electron bifurcation occurs at the NADH-oxidizing bifurcating-flavin adenine dinucleotide (BF-FAD) in EtfA, which has extremely crossed half-potentials and passes the first, high-potential electron to an electron-transferring FAD and via two iron-sulfur clusters eventually to menaquinone. The low-potential electron on the BF-FAD semiquinone simultaneously reduces ferredoxin. We have expressed the genes encodingThermotoga maritimaEtfABCX in E. coli and purified the EtfABCX holoenzyme and the EtfAB subcomplex. The bifurcation activity of EtfABCX was demonstrated by using electron paramagnetic resonance (EPR) to follow accumulation of reduced ferredoxin. To elucidate structural factors that impart the bifurcating ability, EPR and NADH titrations monitored by visible spectroscopy and dye-linked enzyme assays have been employed to characterize four conserved residues, R38, P239, and V242 in EtfA and R140 in EtfB, in the immediate vicinity of the BF-FAD. The R38, P239, and V242 variants showed diminished but still significant bifurcation activity. Despite still being partially reduced by NADH, the R140 variant had no bifurcation activity, and electron transfer to its two [4Fe-4S] clusters was prevented. The role of R140 is discussed in terms of the bifurcation mechanism in EtfABCX and in the other three families of bifurcating enzymes.

IKMTSL-PTE, a Phospholipid-Based EPR Probe for Surface Electrostatic Potential of Biological Interfaces at Neutral pH: Effects of Temperature and Effective Dielectric Constant of the Solvent.

The synthesis and characterization of a lipidlike electrostatic spin probe, (S)-2,3-bis(palmitoyloxy)propyl 2-((4-(4-(dimethylamino)-2-ethyl-1-oxyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-2-yl)benzyl)disulfanyl)ethyl phosphate (IKMTSL-PTE), are being reported. The intrinsic pKa0 of IKMTSL-PTE was determined by X-band (9.5 GHz) electron paramagnetic resonance (EPR) titration of a water-soluble model compound, 4-(dimethylamino)-2-ethyl-2-(4-(((2-hydroxyethyl)disulfanyl)methyl)phenyl)-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl (IKMTSL-ME), an adduct of methanethiosulfonate spin label IKMTSL and 2-mercaptoethanol. The pKa0 of IKMTSL-ME in bulk aqueous solutions was found to be significantly higher than that of 4-(((2-hydroxyethyl)disulfanyl)methyl)-2,2,3,5,5-pentamethylimidazolidin-1-oxyl (IMTSL-ME), an adduct of the corresponding methanethiosulfonate spin label IMTSL and 2-mercaptoethanol (17 °C, pKa0 = 6.16 ± 0.03 vs 20 °C, pKa0 = 3.33 ± 0.03, respectively). A series of EPR titration experiments with IKMTSL-ME in aqueous solutions containing 0-60% v/v isopropanol have been carried out at 17 and 48 °C to determine the effects of temperature and bulk dielectric permittivity constant, ε, on the probe pKa. A linear relationship between the probe pKa and ε has been established and found to be essentially the same at 17 and 48 °C. The polarity term contributing to the pKa of IKMTSL-PTE at an uncharged lipidlike interface was determined by incorporating the probe into electrically neutral micelles formed from nonionic detergent Triton X-100, and it was found, similar to IMTSL-PTE, to be negative. In negatively charged DMPG lipid bilayers, IKMTSL-PTE exhibits ionization transitions with significantly higher pKa values than those previously reported for IMTSL-PTE (e.g., at 17 °C, pKai = 7.80 ± 0.03 vs pKa0 = 5.70 ± 0.05). The surface electrostatic potentials of DMPG lipid bilayers calculated using IKMTSL-PTE titration data were found to be somewhat lower than those calculated using IMTSL-PTE. The lower values measured by IKMTSL-PTE are the likely consequences of the structure of the linker that positions the reporter nitroxide further away from the bilayer plane into aqueous phase. Overall, the ionization transitions of IKMTSL-PTE with pKa values close to the neutral pH range make this lipidlike molecule a valuable spectroscopic EPR probe for studying the electrostatic phenomena at biological interfaces, including lipid bilayer/membrane protein systems, that could be unstable in the acidic pH range accessible by the previously available probes.

Effect of Solution Ionic Strength on the pKa of the Nitroxide pH EPR Probe 2,2,3,4,5,5-Hexamethylimidazolidin-1-oxyl.

Spin probe and spin labeling Electron Paramagnetic Resonance methods are indispensable research tools for solving a wide range of bioanalytical problems-from measuring microviscosity and polarity of phase-separated liquids to oxygen concentrations in tissues. One of the emerging uses of spin probes are the studies of proton transfer-related and surface electrostatic phenomena. The latter Electron Paramagnetic Resonance methods rely on molecular probes containing an additional functionality capable of reversible ionization (protonation, in particular) in the immediate proximity to an Electron Paramagnetic Resonance-active reporter group, such as (N-O•) for nitroxides. The consequent formation of protonated and nonprotonated nitroxide species with different magnetic parameters (A iso, g iso) could be readily distinguished by Electron Paramagnetic Resonance. Bioanalytical Electron Paramagnetic Resonance studies employing pH-sensitive paramagnetic probes typically involve determination of the equilibrium constant (pK a) between the protonated and nonprotonated forms of the nitroxide. However, any chemical equilibrium involving charged species, such as ionization of acids and bases, and so the reversible protonation of the nitroxide, is known to be affected by an ionic strength of the solution. Currently, only scarce data for the effect of the solution ionic strength on the experimental pK a's of the ionizable nitroxides can be found in the literature. Here we have carried out a series of Electron Paramagnetic Resonance titration experiments for aqueous solutions of 2,2,3,4,5,5-hexamethylimidazolidin-1-oxyl (HMI) nitroxide known for one of the largest differences in the isotropic nitrogen hyperfine coupling constant A iso between the protonated and nonprotonated forms. Electrolyte concentration was varied over an exceptionally large range (i.e., from 0.05 to 5.0 M) to elucidate the effect of ionic strength on the ionization constant of this pH-sensitive Electron Paramagnetic Resonance probe and the data were compared to the Debye-Hückel limiting law. Effects of the ionic strength on the magnetic parameters of the ionizable nitroxides are also discussed.

Interfacial Electrostatic Properties of Hydrated Mesoporous and Nanostructured Alumina Powders by Spin Labeling EPR.

Acid-base equilibria and interfacial electrostatic properties of hydrated mesoporous and nanostructured alumina powders are determining factors for the use of these materials in heterogeneous catalysis and as a sorption media for filtration and chromatographic applications including life sciences. Here spin probe electron paramagnetic resonance spectroscopy of pH-sensitive nitroxides was employed to evaluate the surface charge and interfacial acid-base equilibria at the pore surface of mesoporous powders of α-Al2O3, γ-Al2O3, Al2O3 × nH2O, and basic γ-Al2O3 and nanostructured Al2O3 in the form of pristine materials and modified with aluminum-tri-sec-butoxide, hydroxyaluminum glycerate, and several phospholipids. A new pH-sensitive nitroxide probe, 4-dimethylamino-5,5-dimethyl-2-(4-(chloromethyl)phenyl)-2-ethyl-2,5-dihydro-1H-imidazol-1-oxyl hydrochloride semihydrate (nitroxide R1), has been synthesized and characterized. It was found that conditions of preparation of alumina powders exert strikingly large effects on the apparent pK a of nitroxides measured from electron paramagnetic resonance titration curves. Specifically, while the electron paramagnetic resonance titrations curves for the nitroxide R1 in mesoporous powders prepared from basic γ-Al2O3 and Al2O3 × nH2O were shifted by ΔpK a≈ +0.6 and up to ≈ +1.2 pH units respectively, the shift for γ-Al2O3 was found to be much higher: ΔpK a = +3.5. Assuming approximately the same ∆pH = 0.5-1.0 arising from a difference in the hydrogen ion activity between the bulk solution phase and that in a confined pore volume, the samples were ranked in the following order of descending magnitude of the effective surface electrostatic potential Ψ: mesoporous γ-Al2O3 > Al2O3 × nH2O > basic γ-Al2O3 > α-Al2O3. Conditions of the Al2O3 synthesis as well as the surface modification procedures were found to have profound effects on the interfacial electrostatic properties of hydrated samples that are likely related to the nature and concentration of the active sites on the alumina surfaces.

Formation of Ground State Triplet Diradicals from Annulated Rosarin Derivatives by Triprotonation.

Annulated rosarins, β,β'-bridged hexaphyrin(1.0.1.0.1.0) derivatives 1-3, are formally 24 π-electron antiaromatic species. At low temperature, rosarins 2 and 3 are readily triprotonated in the presence of trifluoroacetic acid in dichloromethane to produce ground state triplet diradicals, as inferred from electron paramagnetic resonance (EPR) spectral studies. From an analysis of the fine structure in the EPR spectrum of triprotonated rosarin H₃3(3+), a distance of 3.6 Å between the two unpaired electrons was estimated. The temperature dependence of the singlet-triplet equilibrium was determined by means of an EPR titration. Support for these experimental findings came from calculations carried out at the (U)B3LYP/6-31G* level, which served to predict a very low-lying triplet state for the triprotonated form of a simplified model system 1.

Different Modes of Carbon Monoxide Binding to Acetyl-CoA Synthase and the Role of a Conserved Phenylalanine in the Coordination Environment of Nickel

Acetyl-CoA synthase (ACS) catalyzes the reversible condensation of CO and CH3 units at a unique Ni-Fe cluster, the A cluster, to form an acetyl-Ni intermediate that subsequently reacts with CoA to produce acetyl-CoA. ACS is a component of the multienzyme complex acetyl-CoA decarbonylase/synthase (ACDS) in Archaea and CO dehydrogenase/ACS (CODH/ACS) in bacteria; in both systems, intraprotein CO channeling takes place between the CODH and ACS active sites. Previous studies indicated that protein conformational changes control the chemical reactivity of the A cluster and suggested the involvement of a conserved Phe residue that moves concomitantly into and out of the coordination environment of Ni. Herein, steady-state rate measurements in which both CO and CH3-corrinoid are varied, and rapid methylation reactions of the ACDS β subunit, measured by stopped-flow methods, provide a kinetic model for acetyl-CoA synthesis that includes a description of the inhibitory effects of CO explained by competition of CO and CH3 for the same form of the enzyme. Electron paramagnetic resonance titrations revealed that the formation of a paramagnetic Ni(+)-CO species does not match the kinetics of CO interaction as a substrate but instead correlates well with an inhibited state of the enzyme, which requires revision of previous models that postulate that this species is an intermediate. Characterization of the β subunit F195A variant showed markedly increased substrate reactivity with CO, which provides biochemical functional evidence of steric shielding of the CO substrate interaction site by the phenyl group side chain. The phenyl group also likely enhances the nucleophilicity of the Ni center to facilitate CH3 group transfer. A model was developed for how the catalytic properties of the A cluster are optimized by linking conformational changes to a repositionable aromatic shield able to modulate the nucleophilicity of Ni, sterically select the most productive order of substrate addition, and overcome intrinsic inhibition by CO.

Tetrathiafulvalene Hetero Radical Cation Dimerization in a Redox-Active [2]Catenane

The electronic properties of tetrathiafulvalene (TTF) can be tuned by attaching electron-donating or electron-withdrawing substituents. An electron-rich macrocyclic polyether containing two TTF units of different constitutions, namely 4,4'-bis(hydroxymethyl)tetrathiafulvalene (OTTFO) and 4,4'-bisthiotetrathiafulvalene (STTFS), has been synthesized. On two-electron oxidation, a hetero radical dimer is formed between OTTFO(•+) and STTFS(•+). The redox behavior of the macrocyclic polyether has been investigated by electrochemical techniques and UV-vis and electron paramagnetic resonance (EPR) spectroscopies. The [2]catenane in which the macrocyclic polyether is mechanically interlocked with the cyclobis(paraquat-p-phenylene) (CBPQT(4+)) ring has also been prepared using template-directed protocols. In the case of the [2]catenane, the formation of the TTF hetero radical dimer is prevented sterically by the CBPQT(4+) ring. After a one-electron oxidation, a 70:30 ratio of OTTFO(•+) to STTFS(•+) is present at equilibrium, and, as a result, two translational isomers of the [2]catenane associated with these electronically different isomeric states transpire. EPR titration spectroscopy and simulations reveal that the radical states of the two constitutionally different TTF units in the [2]catenane still experience long-range electronic intramolecular coupling interactions, despite the presence of the CBPQT(4+) ring, when one or both of them are oxidized to the radical cationic state. These findings in the case of both the free macrocyclic polyether and the [2]catenane have led to a deeper fundamental understanding of the mechanism of radical cation dimer formation between constitutionally different TTF units.

Iron(III)- and copper(II) complexes of an asymmetric, pentadentate salen-like ligand bearing a pendant carboxylate group

The equilibrium and solution structural properties of the iron(III) and copper(II) complexes of an asymmetric salen-like ligand (N,N′-bis(2-hydroxybenzyl)-2,3-diamino-propionic acid, H3bhbdpa) bearing a pendant carboxylate group were characterized in aqueous solution by potentiometric, pH-dependent electron paramagnetic resonance (EPR) and UV–Vis (UV–Visible) measurements. In the equimolar systems the pentadentate ligand forms very stable, differently protonated mononuclear complexes with both metal ions. In the presence of iron(III) {NH, PhO−, COO−}, {2NH, 2PhO−, COO−} and {2NH, 2PhO−, COO−, OH−} coordinated complexes are dominant. The EPR titrations reflected the presence of microscopic complex formation pathways, leading to the formation of binding isomers in case of Cu(H2bhbdpa)+, Cu(Hbhbdpa) and Cu(bhbdpa)−. The {2NH, 2PhO−+COO−/H2O} coordinated Cu(bhbdpa) is the only species between pH 6–11. At twofold excess of metal ion dinuclear complexes were detected with both iron(III) and copper(II). In presence of iron(III) a μ-carboxylato-μ-hydroxo-bridged dinuclear complex (Fe2(bhbdpa)(OH)3) is formed from Fe(H2bhbdpa)2+ through overlapping proton release processes, providing one of the rare examples for the stabilization of an endogenous carboxylate bridged diiron core in aqueous solution. The complex Cu2(bhbdpa)+ detected in the presence of copper(II) is a paramagnetic (S=1) species with relatively weakly coupled metal ions.

Inclusion Complexes of PBN-Type Nitrone Spin Traps and Their Superoxide Spin Adducts with Cyclodextrin Derivatives: Parallel Determination of the Association Constants by NMR Titrations and 2D-EPR Simulations

(1)H NMR and electron paramagnetic resonance (EPR) titrations were used to determine the association constants of the complexes of alpha-phenyl-N-tert-butylnitrone (PBN) analogues and their superoxide spin adducts, respectively, with methylated beta-cyclodextrins. A 1:1 stoichiometry for the nitrones with randomly methylated beta-cyclodextrin and 2,6-di-O-methyl-beta-cyclodextrin and 1:1 and 1:2 stoichiometries for the corresponding cyclodextrin-nitroxide complexes were observed. After the superoxide radical spin trapping reaction, EPR titrations afforded the association constants of the corresponding cyclodextrin-nitroxide complexes. Two-dimensional EPR simulations indicated a bimodal inclusion of the nitroxide free radical spin adducts into the cyclodextrins. For all the nitrone-cyclodextrin and nitroxide-cyclodextrin complexes, the association constants were always higher for the nitroxide complexes than for the nitrone complexes. A cooperative system concerning the complexation of the nitroxide spin adduct with a cyclodextrin was evidenced by EPR titrations. The efficiency of the cyclodextrin inclusion technique to trap superoxide and to resist bioreduction by sodium l-ascorbate was also investigated.

Oxidation-reduction properties of the ferredoxin-linked glutamate synthase from spinach leaf

Oxidation-reduction titrations have been conducted to determine the midpoint potential ( E m ) values of the three electron-carrying prosthetic groups of the ferredoxin-linked glutamate synthase isolated from spinach leaves. Titrations using electron paramagnetic resonance (EPR) signals to monitor the oxidation state of the [3Fe 4S] +0 cluster found in the enzyme, indicated the presence of a single n = 1 component with E m = −170 mV at pH 7.7. Titrations using absorbance changes in the visible region to monitor the oxidation states of the FAD and FMN groups present in the enzyme could be fit to a single n = 2 Nernst curve with E m = −180 mV at pH 7.7. The magnitude of the absorbance change observed during this titration accounts for all of the FMN and FAD found in the enzyme, indicating that the two flavins are either isopotential or differ in E m by less than about 30 mV. Neither optical nor EPR titrations gave any evidence for the presence of stable flavin free radicals. These results represent the first characterization of the redox properties of the prosthetic groups of a ferredoxin-dependent glutamate synthase.

Oxidation-reduction properties of the ferredoxin-linked glutamate synthase from spinach leaf

Oxidation-reduction titrations have been conducted to determine the midpoint potential ( E m) values of the three electron-carrying prosthetic groups of the ferredoxin-linked glutamate synthase isolated from spinach leaves. Titrations using electron paramagnetic resonance (EPR) signals to monitor the oxidation state of the [3Fe 4S] +0 cluster found in the enzyme, indicated the presence of a single n = 1 component with E m = −170 mV at pH 7.7. Titrations using absorbance changes in the visible region to monitor the oxidation states of the FAD and FMN groups present in the enzyme could be fit to a single n = 2 Nernst curve with E m = −180 mV at pH 7.7. The magnitude of the absorbance change observed during this titration accounts for all of the FMN and FAD found in the enzyme, indicating that the two flavins are either isopotential or differ in E m by less than about 30 mV. Neither optical nor EPR titrations gave any evidence for the presence of stable flavin free radicals. These results represent the first characterization of the redox properties of the prosthetic groups of a ferredoxin-dependent glutamate synthase.

A spectroelectrochemical cell designed for low temperature electron paramagnetic resonance titration of oxygen-sensitive proteins

In this paper we describe an anaerobic titrator made virtually from glass with a small amount of high vacuum epoxy mounted directly to a quartz EPR tube. A complete titration may be carried out with as little as 600 μl of sample. This cell features the anaerobic manipulation of an electrochemically poised solution from an electrochemical pouch to an EPR tube. The cell uses a gold foil working electrode and Ag AgCl reference and counter electrodes. The reference and counter electrodes are isolated from the sample by leached Vycor glass. In the work reported here, we used this cell to determine the equilibrium redox potential of methyl viologen in an EPR titration. With methyl viologen as an indicator we found that the cell has a residual oxygen level of 1.5 μ m with a leak rate of 0.005 nmol/min. After moving the solution into the EPR tube, freezing, performing EPR, and thawing, the potential of the methyl viologen solution drifted only 2 mV. During the titration, the poised potentials were stable, drifting only 1 mV/min. Formal potentials as low as −630 mV in a vitamin B 12-type protein have been determined with this cell (S. R. Harder, W.-P. Lu, B. A. Feinberg, and S. W. Ragsdale (1989) Biochemistry, in press) .

Interaction of spin-labeled nicotinamide adenine dinucleotide phosphate with chicken liver fatty acid synthase

The spatial relationships between the four reduced nicotinamide adenine dinucleotide phosphate (NADPH) binding sites on chicken liver fatty acid synthase were explored with electron paramagnetic resonance (EPR) and spin-labeled analogues of NADP+. The analogues were prepared by reaction of NADP+ with 2,2,5,5-tetramethyl-1-oxy-3-pyrroline-3-carboxylic acid, with 1,1'-carbonyldiimidazole as the coupling reagent. Several esterification products were characterized, and the interaction of the N3' ester of NADP+ with the enzyme was examined in detail. Both 1H13, 14N and 2H13, 15N spin-labels were used: the EPR spectrum was simpler, and the sensitivity greater, for the latter. The spin-labeled NADP+ is a competitive inhibitor of NADPH in fatty acid synthesis, and an EPR titration of the enzyme with the modified NADP+ indicates four identical binding sites per enzyme molecule with a dissociation constant of 124 microM in 0.1 M potassium phosphate and 1 mM ethylenediaminetetraacetic acid (pH 7.0) at 25 degrees C. The EPR spectra indicate the bound spin-label is immobilized relative to the unbound probe. No evidence for electron-electron interactions between bound spin-labels was found with the native enzyme, the enzyme dissociated into monomers, or the enzyme with the enoyl reductase sites blocked by labeling the enzyme with pyridoxal 5'-phosphate. Furthermore, the EPR spectrum of bound ligand was the same in all cases. This indicates that the bound spin-labels are at least 15 A apart, that the environment of the spin-label at all sites is similar, and that the environment is not altered by major structural changes in the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Physical and chemical studies on bacterial superoxide dismutases. Purification and some anion binding properties of the iron-containing protein of Escherichia coli B.

Highly purified iron superoxide dismutase was obtained from Escherichia coli B using a modification of the procedure of Yost and Jridovich (Yost, F. J., Jr., and Fridovich, I. (1973) J. Biol. Chem. 248, 4905-4908). The protein contained 1.8 +/- 0.2 atoms of iron per 38,700 g of protein. We have found that cyanide does not bind to the Fe3+ ion of iron dismutase but fluoride and azide have moderately large binding constants. Optical and electron paramagnetic resonance (EPR) measurements suggested that 2 fluoride ions could associate with each iron atom with the first having an association constant of approximately 520 M-1 and the second with an estimated value of 24 M-1. Activity measurements yielded an inhibition constant for fluoride of 30 M-1. At room temperature only one azide binds to the Fe3+ (K = 760 M-1) and this does not interfere with superoxide dismutase activity. Upon freezing solutions of iron superoxide dismutase in the presence of excess azide their color changes from yellow to pink. Combined EPR and optical titrations with azide suggest the presence of two binding sites on Fe3+ with only the first being occupied at room temperature and the second binding azide only upon freezing the solution. The results suggest that each Fe3+ ion of this superoxide dismutase has two coordination positions available for interaction with solute molecules but only one is necessary for catalysis of the superoxide dismutation reaction. The EPR, optical, and circular dichroism spectra of the native protein and the various fluoride and azide complexes are presented.

Purification and properties of paramagnetic protein from clostridium pasteurianum W5

The purification to homogeneity of the non-heme iron protein, sometimes referred to as either “red protein’ or “paramagnetic protein’, from Clostridium pasteurianum W5 extracts is described and its physicochemical properties studied. This paramagnetic protein ( g  1.94) has a molecular weight of about 25 000 and contains two iron and two acid-labile sulfur atoms per mol of protein. Its midpoint potential at pH 7.5, as determined by electron paramagnetic resonance titration, is —300 mV. Optical circular dichroism and electron paramagnetic resonance spectra of the paramagnetic protein are similar to those of two iron-two acid-labile sulfur ferredoxins. The biochemical reduction of the purified protein was also studied.

Potentiometric titration of soluble and bound iron-sulfur proteins monitored by circular dichroism and electron paramagnetic resonance spectroscopy

Potentiometric titrations of iron-sulfur proteins monitored by changes in circular dichroism (CD) or electron-paramagnetic-resonance (EPR) spectrum are presented. Both monitoring techniques have the distinct advantage that interferences caused by the absorption changes of the redox mediators during a titration, which limit the use of the absorption spectrophotometry for monitoring, are either not monitored, or do not represent a serious interference. The CD tritration is illustrated with soluble spinach ferrodoxin and the EPR titration with bound iron-sulfur proteins of green plant photosystem I. The anacrobic titration apparatus and other experimental considerations are described in detail.

Electron Paramagnetic Resonance and Proton Relaxation Rate Studies of Spin-labeled Creatine Kinase and Its Complexes

Abstract A spin-labeled derivative of rabbit muscle creatine kinase has been prepared by reaction of the essential sulfhydryl group at the active sites with a stoichiometric amount of the nitroxide radical N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) iodoacetamide. The spin-labeled enzyme retains the ability to bind nucleotide substrates although it lacks the phosphotransferase and ATPase activities of the native enzyme. Two magnetic resonance properties of the specifically spin-labeled enzyme and its substrate complexes were used to probe the local environment of the covalently bound radical: (a) the electron paramagnetic resonance (EPR) spectrum and (b) the enhancement of the proton relaxation rate of water protons. The protein-bound radical shows a broad, asymmetric EPR spectrum resembling a solid state powder spectrum; the outer peaks are separated by 62 gauss compared with 31 gauss for the free radical and the amplitude of the central peak is 14 times greater in the unbound than the bound spin label. In addition, the spin-labeled protein enhances the water proton relaxation rate by approximately 1 order of magnitude. Both phenomena indicate a high degree of immobilization of the bound spin label. Metal nucleotide binding increases immobilization of the spin label as evidenced by the EPR spectrum. The spectral changes represent the first direct evidence for a proposed conformational change in the region of the active site on addition of divalent ions to the enzyme-nucleotide binary complex. The proton relaxation rate of water is even more sensitive to local structural changes and reveals differences between the enzyme and its binary nucleotide complexes as well as between binary and ternary enzyme-metal nucleotide complexes. Creatine alone or in combination with metal nucleotides fails to affect either the EPR spectrum or the water proton relaxation rate of the spin-labeled enzyme. The nucleotide complexes of the alkali earth cations, Mg(II), Ca(II), Sr(II), and Ba(II), produce approximately the same changes in the EPR of the spin-labeled enzyme, amounting to a diminution of 20% in central peak amplitude upon addition of metal-ADP and a somewhat smaller effect with the ATP complexes. Quantitative differences in the properties of the different metal complexes are most easily observed by measurement of the proton relaxation rate of water under optimal temperature and frequency conditions. The ternary complexes of the irreversibly inhibitory Zn(II) nucleotides with spin-labeled enzyme have qualitatively different EPR spectra than do the corresponding complexes of alkali earth ions. Apparent dissociation constants have been calculated from both EPR and proton relaxation rate titrations for the dissociation of the alkali earth nucleotides from the ternary complexes. All are of the same order of magnitude as for the native enzyme, about 0.1 mm for ADP ternary complexes.

Studies on Cytochrome c Peroxidase: XVII. STOICHIOMETRY AND MECHANISM OF THE REACTION OF COMPOUND ES WITH DONORS

Abstract The stoichiometry of the reaction of cytochrome c peroxidase with hydrogen peroxide, and of the resulting Compound ES with both ferrocyanide and ferrocytochrome c as donor has been re-examined in detail over a wide pH range. It was confirmed that cytochrome c peroxidase and hydrogen peroxide react in a 1:1 molar ratio, and that 2 molecules of either donor are required to reduce Compound ES completely. The re-examination of the change in absorbance on formation of Compound ES gave a more reliable value of the difference extinction coefficient than those previously reported. It was established that the reaction of Compound ES with donors occurs in at least two stages of comparable rates. Comparison of optical and electron paramagnetic resonance titration curves showed that the two oxidizing equivalents of Compound ES were associated with distinct and partially independent changes in the properties of the enzyme. The rate of reaction of Compound ES with ferrocyanide was measured over the pH range 5 to 8, and the reaction was found to be biphasic. Several mechanisms were considered to account for these observations. All of the mechanisms involve an enzyme intermediate compound which is only one oxidizing equivalent above the native state. A theoretical analysis of the observed kinetics and titration curves showed that the data presently available do not unequivocally distinguish among all the proposed mechanisms. However, they strongly support a mechanism in which two one-oxidizing-equivalent intermediates are at rapid equilibrium.