Tryptophan-based radical in the catalytic mechanism of versatile peroxidase from Bjerkandera adusta

Versatile peroxidase (VP) from Bjerkandera adusta is a structural hybrid between lignin (LiP) and manganese (MnP) peroxidase. This hybrid combines the catalytic properties of the two above peroxidases, being able to oxidize typical LiP and MnP substrates. The catalytic mechanism is that of classical peroxidases, where the substrate oxidation is carried out by a two-electron multistep reaction at the expense of hydrogen peroxide. Elucidation of the structures of intermediates in this process is crucial for understanding the mechanism of substrate oxidation. In this work, the reaction of H(2)O(2) with the enzyme in the absence of substrate has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical is approximately 30%. Progressive microwave power saturation measurements indicate that the radical is weakly coupled to a paramagnetic metal ion, suggesting an amino acid radical in moderate distance from the ferryl heme. A tryptophan radical was identified as a protein-based radical formed during the catalytic mechanism of VP from Bjerkandera adusta through X-band and high-field EPR measurements at 94 GHz, aided by computer simulations for both frequency bands. A close analysis of the theoretical model of the VP from Bjerkandera sp. shows the presence of a tryptophan residue near to the heme prosthetic group, which is solvent-exposed as in the case of LiP and other VPs. The catalytic role of this residue in a long-range electron-transfer pathway is discussed.     

Kinetics and energetics of intramolecular electron transfer in yeast cytochrome c peroxidase

The oxidation of ferric cytochrome c peroxidase by hydrogen peroxide yields a product, compound ES [Yonetani, T., Schleyer, H., Chance, B., & Ehrenberg, A. (1967) in Hemes and Hemoproteins (Chance, B., Estabrook, R. W., & Yonetani, T., Eds.) p 293, Academic Press, New York], containing an oxyferryl heme and a protein free radical [Dolphin, D., Forman, A., Borg, D. C., Fajer, J., & Felton, R. H. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 614-618]. The same oxidant takes the ferrous form of the enzyme to a stable Fe(IV) peroxidase [Ho, P. S., Hoffman, B. M., Kang, C. H., & Margoliash, E. (1983) J. Biol. Chem. 258, 4356-4363]. It is 1 equiv more highly oxidized than the ferric protein, contains the oxyferryl heme, but leaves the radical site unoxidized. Addition of sodium fluoride to Fe(IV) peroxidase gives a product with an optical spectrum similar to that of the fluoride complex of the ferric enzyme. However, reductive titration and electron paramagnetic resonance (EPR) data demonstrate that the oxidizing equivalent has not been lost but rather transferred to the radical site. The EPR spectrum for the radical species in the presence of Fe(III) heme is identical with that of compound ES, indicating that the unusual characteristics of the radical EPR signal do not result from coupling to the heme site. By stopped-flow measurements, the oxidizing equivalent transfer process between heme and radical site is first order, with a rate constant of 0.115 s-1 at room temperature, which is independent of either ligand or protein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heme spin states and peroxide-induced radical species in prostaglandin H synthase

We have examined the optical, magnetic circular dichroism, and electron paramagnetic resonance (EPR) spectra of pure ovine prostaglandin H synthase in its resting (ferric) and ferrous states and after addition of hydrogen peroxide or 15-hydroperoxyeicosatetraenoic acid. In resting synthase, the distribution of heme between high- and low-spin forms was temperature-dependent: 20% of the heme was low-spin at room temperature whereas 50% was low-spin at 12 K. Two histidine residues were coordinated to the heme iron in the low-spin species. Anaerobic reduction of the synthase with dithionite produced a high-spin ferrous species that had no EPR signals. Upon reaction with the resting synthase, both hydroperoxides quickly generated intense (20-40% of the synthase heme) and complex EPR signals around g = 2 that were accompanied by corresponding decreases in the intensity of the signals from ferric heme at g = 3 and g = 6. The signal generated by HOOH had a doublet at g = 2.003, split by 22 G, superimposed on a broad component with a peak at g = 2.085 and a trough at g = 1.95. The lipid hydroperoxide generated a singlet at g = 2.003, with a linewidth of 25 G, superimposed on a broad background with a peak at g = 2.095 and a trough around g = 1.9. These EPR signals induced by hydroperoxide may reflect synthase heme in the ferryl state complexed with a free radical derived from hydroperoxide or fragments of hydroperoxide.     

The detection of two electron paramagnetic resonance radical signals associated with chloroperoxidase compound I

The electron paramagnetic resonance spectra of chloroperoxidase Compound I and native enzyme are compared. Upon the formation of Compound I, the g = 2.62, 2.26, and 1.82 signals associated with native enzyme disappear and are replaced by two new EPR signals, a sharp signal at g = 2.008 and a broad signal at g = 1.73. The g = 2.008 signal accounts for only 2% of the theoretical spins while the broad signal at g = 1.73 accounts for 60 to 70% of the theoretical spins in Compound I. The g = 1.73 broad signal is reminiscent of the broad EPR signal associated with horseradish peroxidase Compound I. however, the chloroperoxidase Compound I signal has a significantly different g value. The results suggest that the g = 1.73 signal represents a porphyrin pi cation radical which has a stronger coupling to the heme ferryl iron than is the case with horseradish peroxidase Compound I.     

Electron paramagnetic resonance characterization of tyrosine radical, M+, in site-directed mutants of photosystem II(t).

Photosystem II contains two well-characterized tyrosine radicals, D(.) and Z(.). Z is an electron carrier between the primary chlorophyll donor and the manganese catalytic site and is essential for enzymatic function. On the other hand, D forms a stable radical with no known role in oxygen evolution. D(.) and Z(.) give rise to similar, but not identical, room temperature electron paramagnetic resonance (EPR) signals, which can be distinguished by their decay kinetics. A third room temperature EPR signal has also been observed in site-directed mutants in which a nonredox active amino acid is substituted at the D or Z site. This four-line EPR signal has been shown to have a tyrosine origin by isotopic labeling (Boerner and Barry, 1994, J. Biol. Chem. 269:134-137), but such an EPR signal has never before been observed from a tyrosyl radical. The radical giving rise to this third unique signal has been named M+. Here we provide kinetic evidence that this signal arises from a third redox active tyrosine, distinct from tyrosine D and Z, in the photosystem II reaction center. Isotopic labeling and EPR spectroscopy provide evidence that M is a covalently modified tyrosine.     

Powder and single-crystal electron paramagnetic resonance studies of yeast cytochrome c peroxidase and its peroxide and its peroxide compound, Compound ES

Electron paramagnetic resonance absorption spectrum of ferric cytochrome c peroxidase exhibited a mixture of high- and low-spin compounds. The principal values and the eigenvectors of the g-tensor for the low-spin species were determined by single-crystal EPR spectroscopy at 77 K. The powder EPR spectra of the peroxide compound, Compound ES, were measured at S-, X-, and Q-band microwave frequencies. Careful examination at 77 K showed a narrow free radical-like signal at g = 2.004 with hyperfine structures accompanied by a broad signal spreading on both low- and high-field sides. Single-crystal EPR analyses of Compound ES clearly demonstrated that there exist at least two different radical species: one is isotropic with hyperfine structure at g = 2.004 and the other exhibits an axially symmetric signal at 5 K and broad signal centered at g = 2.004 at 77 K, respectively. The principal values and the eigenvectors of the g-tensor for the axially symmetric signal were determined: g(parallel) = 2.034 and g(perpendicular) = 2.006, 1.999. The orientation of the unique axis (g(parallel)) was found to be identical to that of the heme normal. A new radical signal with complicated hyperfine structures in the g = 2.004 region was observed upon illumination of Compound ES at both 5 and 77 K. The photoinduced species grew effectively by the illumination light around 500 nm. On warming to -80 degrees C, the photoinduced signal was reversibly brought back to the original radical species of Compound ES via an intermediate species. From these results, we have proposed the possible sites for the free radical centers in Compound ES.     

EPR and optical changes of the photosystem II reaction center produced by low temperature illumination

Electron paramagnetic resonance (EPR) and absorption spectroscopy have been used to study the low temperature photochemical behavior of the Photosystem II D-1/D-2/ cytochrome b559 reaction center complex. The reaction center displays large triplet state EPR signals which are attenuated after actinic illumination at low temperatures in the presence of sodium dithionite. Concomitant with the triplet attenuation is the buildup of a structured radical signal with an effective g value of 2.0046 and a peak-to-peak width of 11.9 G. The structure in the signal is suggestive of it being comprised in part of the anion radical of pheophytin a. This assignment is corroborated by low temperature optical absorbance measurements carried out after actinic illumination at the low temperatures which show absorption bleachings at 681 nm, 544 nm and 422 nm and an absorbance buildup at 446 nm indicating the formation of reduced pheophytin.Abbreviations EPR electron paramagnetic resonance     

Chromaffin granule membranes contain at least three heme centers: direct evidence from EPR and absorption spectroscopy

Low-temperature electron paramagnetic resonance (EPR) spectroscopy, circular dichroism and two-component redox titration have previously provided evidence for two different ascorbate-reducible heme centers in cytochrome b(561) present in chromaffin granule membranes. These species have now been observed by room and liquid nitrogen temperature absorption spectroscopy. The visualization of these heme centers becomes possible as a consequence of utilizing chromaffin granule membranes prepared by a mild procedure. Additionally, a new redox center, not reducible by ascorbate, was discovered by both EPR and absorption spectroscopy. It constitutes about 15% of the heme absorbance of chromaffin membranes at 561 nm and has EPR characteristics of a well-organized highly axial low-spin heme center (thus making it unlikely that it is a denatured species). This species is either an alternative form of one of the hemes of cytochrome b(561) that has a very low redox potential or a b-type cytochrome distinct from b(561).     

Direct evidence for a tyrosine radical in the reaction of cytochrome c oxidase with hydrogen peroxide.

Cytochrome c oxidase (COX) catalyzes the reduction of oxygen to water, a process which is accompanied by the pumping of four protons across the membrane. Elucidation of the structures of intermediates in these processes is crucial for understanding the mechanism of oxygen reduction. In the work presented here, the reaction of H(2)O(2) with the fully oxidized protein at pH 6.0 has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical based on comparison with other paramagnetic centers in COX was approximately 20%. Recent crystallographic data have shown that one of the Cu(B) ligands, His 276 (in the bacterial case), is cross-linked to Tyr 280 and that this cross-linked tyrosine is ideally positioned to participate in dioxygen activation. Here selectively deuterated tyrosine has been incorporated into the protein, and a drastic change in the line shape of the EPR signal observed above has been detected. This would suggest that the observed EPR signal does indeed arise from a tyrosine radical species. It would seem also quite possible that this radical is an intermediate in the mechanism of oxygen reduction.     

EPR studies of the water oxidizing complex in the S1 and the higher S states: the manganese cluster and Y(Z) radical

The parallel polarization electron paramagnetic resonance (EPR) method has been applied to investigate manganese EPR signals of native S1 and S3 states of the water oxidizing complex (WOC) in photosystem (PS) II. The EPR signals in both states were assigned to thermally excited states with S=1, from which zero-field interaction parameters D and E were derived. Three kinds of signals, the doublet signal, the singlet-like signal and g=11-15 signal, were detected in Ca2+-depleted PS II. The g=11-15 signal was observed by parallel and perpendicular modes and assigned to a higher oxidation state beyond S2 in Ca2+-depleted PS II. The singlet-like signal was associated with the g=11-15 signal but not with the Y(Z) (the tyrosine residue 161 of the D1 polypeptide in PS II) radical. The doublet signal was associated with the Y(Z) radical as proved by pulsed electron nuclear double resonance (ENDOR) and ENDOR-induced EPR. The electron transfer mechanism relevant to the role of Y(Z) radical was discussed.     

Effect of temperature on the electron paramagnetic resonance spectrum of irradiated alanine.

Polycrystalline samples of the amino acid L-alpha-alanine have been irradiated with X rays at both room temperature and higher temperatures. The electron paramagnetic resonance (EPR) spectra of alanine powder irradiated at room temperature are dominated by the well-known room-temperature-stable alanine radical CH3C*HCOOH. Upon heating of room-temperature-irradiated alanine powder, a strong decay of the signal was observed, and the features of the spectrum recently ascribed to a second stable radical in alanine irradiated at room temperature become more pronounced, providing an experimental isolation of this second alanine radical. In combination with the high-temperature experiments, a multivariate statistical decomposition method, maximum likelihood common factor analysis, was used to determine the number of components in irradiated alanine powder which behave differently as a function of temperature. The EPR components found in the present study are compared with simulations using earlier EPR and ENDOR single-crystal data.     

Chloroperoxidase compound I: Electron paramagnetic resonance and M?ssbauer studies

The green primary compound of chloroperoxidase was prepared by freeze-quenching the enzyme after rapid mixing with a 5-fold excess of peracetic acid. The electron paramagnetic resonance (EPR) spectra of these preparations consisted of at least three distinct signals that could be assigned to native enzyme, a free radical, and the green compound I as reported earlier. The absorption spectrum of compound I was obtained through subtraction of EPR signals measured under passage conditions. The signal is well approximated by an effective spin Seff = 1/2 model with g = 1.64, 1.73, 2.00 and a highly anisotropic line width. M?ssbauer difference spectra of compound I samples minus native enzyme showed well-resolved magnetic splitting at 4.2 K, an isomer shift delta Fe = 0.15 mm/s, and quadrupole splitting delta EQ = 1.02 mm/s. All data are consistent with the model of an exchange-coupled spin S = 1 ferryl iron and a spin S' = 1/2 porphyrin radical. As a result of the large zero field splitting, D, of the ferryl iron and of intermediate antiferromagnetic exchange, S.J.S'.J approximately 1.02 D, the system consists of three Kramers doublets that are widely separated in energy. The model relates the EPR and M?ssbauer spectra of the ground doublet to the intrinsic parameters of the ferryl iron, D/k = 52 K, E/D congruent to 0.035, and A perpendicular (gn beta n) = 20 T, and the porphyrin radical.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tryptophan or tyrosine? On the nature of the amino acid radical formed following hydrogen peroxide treatment of cytochrome c oxidase

It has been reported that different amino acid radicals are formed following the addition of hydrogen peroxide to cytochrome c oxidase (CcO) from bovine heart or from Paracoccus denitrificans. A broad unresolved signal in the electron paramagnetic resonance (EPR) spectra of bovine CcO has been assigned to a tryptophan radical, probably Trp126 [Rigby et al. Biochemistry 2000, 39, 5921-5928]. In the P. denitrificans enzyme, a similarly broad signal but with a well-resolved hyperfine structure was shown to originate from a tyrosyl radical and was tentatively assigned to the active site Tyr280 [MacMillan et al. Biochemistry 1999, 38, 9179-9184]. We confirm that the EPR signal from P. denitrificans CcO can be simulated using spectral parameters typical for known Tyr radicals in other systems. However, the rotational conformation of the phenolic ring of Tyr280 is inconsistent with our simulation. Instead, the simulation parameters we used correspond to the rotational conformation of ring that matches very accurately the conformation found in Tyr167, a residue that is close enough ( approximately 10 A) to the binuclear centre to readily donate an electron. The broad unresolved EPR signal in the bovine oxidase has been thought previously to be inconsistent with a tyrosyl radical. However, we have simulated a hypothetical EPR spectrum arising from a Tyr129 radical (the equivalent of Tyr167 in P. denitrificans CcO) and showed that it is similar to the observed broad signal. The possibility exists, therefore, that the homological tyrosine amino acid (Tyr167/Tyr129) is responsible for the EPR spectrum in both the Paraccoccus and the bovine enzyme. This correspondence between the two enzymes at least allows the possibility that this radical may have functional importance.     

Chemical nature of the porphyrin pi cation radical in horseradish peroxidase compound I

The electron paramagnetic resonance (EPR) and M?ssbauer properties of native horseradish peroxidase have been compared with those of a synthetic derivative of the enzyme in which a mesohemin residue replaces the natural iron protoporphyrin IX heme prosthetic group. The oxyferryl pi cation radical intermediate, compound I, has been formed from both the native and synthetic enzyme, and the magnetic properties of both intermediates have been examined. The optical absorption characteristics of compound I prepared from mesoheme-substituted horseradish peroxidase are different from those of the compound I prepared from native enzyme [DiNello, R. K., & Dolphin, D. (1981) J. Biol. Chem. 256, 6903-6912]. By analogy to model-compound studies, it has been suggested that these optical absorption differences are due to the formation of an A2u and an A1u pi cation radical species, respectively. However, the EPR and M?ssbauer properties of the native and synthetic enzyme and of their oxidized intermediates are quite similar, if not identical, and the data favor an A2u radical for both compounds I.     

Identification and characterization of tyrosyl radical formation in Mycobacterium tuberculosis catalase-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.     

Comparative study of tyrosine radicals in hemoglobin and myoglobins treated with hydrogen peroxide

The reactions of hydrogen peroxide with human methemoglobin, sperm whale metmyoglobin, and horse heart metmyoglobin were studied by electron paramagnetic resonance (EPR) spectroscopy at 10 K and room temperature. The singlet EPR signal, one of the three signals seen in these systems at 10 K, is characterized by a poorly resolved, but still detectable, hyperfine structure that can be used to assign it to a tyrosyl radical. The singlet is detectable as a quintet at room temperature in methemoglobin with identical spectral features to those of the well characterized tyrosyl radical in photosystem II. Hyperfine splitting constants found for Tyr radicals were used to find the rotation angle of the phenoxyl group. Analysis of these angles in the crystal structures suggests that the radical resides on Tyr151 in sperm whale myoglobin, Tyr133 in soybean leghemoglobin, and either alphaTyr42, betaTyr35, or betaTyr130 in hemoglobin. In the sperm whale metmyoglobin Tyr103Phe mutant, there is no detectable tyrosyl radical present. Yet the rotation angle of Tyr103 (134 degrees) is too large to account for the observed EPR spectrum in the wild type. Tyr103 is the closest to the heme. We suggest that Tyr103 is the initial site of the radical, which then rapidly migrates to Tyr151.     

Characterization of a novel g' = 2.95 EPR signal from the binuclear center of mitochondrial cytochrome c oxidase.

The oxidized binuclear heme a3/CuB center of slow forms of bovine cytochrome oxidase exhibits a characteristic EPR signal at g' = 12. Following the (rapid) dithionite reduction of heme a and CuA, an additional EPR signal becomes apparent at g' = 2.95. As electrons enter the binuclear center this signal decays at the same slow rate as the g' = 12 signal. In the fully oxidized slow enzyme the small g' = 2.95 signal is usually masked by the g = 3 heme a signal, but it is readily detectable at low temperatures and high microwave powers. It is present in both the intrinsic and formate-ligated slow enzymes, but not in any form of fast preparation. The g' = 2.95 signal has similar temperature dependence and microwave power saturation characteristics to the g' = 12 signal. We conclude that the signal arises from the same population of binuclear centers responsible for the g' = 12 signal. The appearance of a signal at g' = 2.95 in X-band EPR is consistent with, but does not prove, the model of Hagen where the g' = 12 signal arises from a ferryl heme a3, with CuB cuprous and EPR-silent (Hagen, W. R. (1982) Biochim. Biophys. Acta 708, 82-98).     

Angular dependences of perpendicular and parallel mode electron paramagnetic resonance of oxidized beef heart cytochrome c oxidase

Cytochrome c oxidase catalyzes the reduction of oxygen to water with a concomitant conservation of energy in the form of a transmembrane proton gradient. The enzyme has a catalytic site consisting of a binuclear center of a copper ion and a heme group. The spectroscopic parameters of this center are unusual. The origin of broad electron paramagnetic resonance (EPR) signals in the oxidized state at rather low resonant field, the so-called g' = 12 signal, has been a matter of debate for over 30 years. We have studied the angular dependence of this resonance in both parallel and perpendicular mode X-band EPR in oriented multilayers containing cytochrome c oxidase to resolve the assignment. The "slow" form and compounds formed by the addition of formate and fluoride to the oxidized enzyme display these resonances, which result from transitions between states of an integer-spin multiplet arising from magnetic exchange coupling between the five unpaired electrons of high spin Fe(III) heme a(3) and the single unpaired electron of Cu(B). The first successful simulation of similar signals observed in both perpendicular and parallel mode X-band EPR spectra in frozen aqueous solution of the fluoride compound of the closely related enzyme, quinol oxidase or cytochrome bo(3), has been reported recently (Oganesyan et al., 1998, J. Am. Chem. Soc. 120:4232-4233). This suggested that the exchange interaction between the two metal ions of the binuclear center is very weak (|J| approximately 1 cm(-1)), with the axial zero-field splitting (D approximately 5 cm(-1)) of the high-spin heme dominating the form of the ground state. We show that this model accounts well for the angular dependences of the X-band EPR spectra in both perpendicular and parallel modes of oriented multilayers of cytochrome c oxidase derivatives and that the experimental results are inconsistent with earlier schemes that use exchange coupling parameters of several hundred wavenumbers.     

Modifications in heme iron of free and vesicle bound cytochrome c by tert-butyl hydroperoxide: a magnetic circular dichroism and electron paramagnetic resonance investigation

To characterize changes to the heme and the influence of membrane lipids in the reaction of cytochrome c with peroxides, we studied the reaction of cytochrome c with tert-butyl hydroperoxide (tert-BuOOH) by magnetic circular dichroism (MCD) and direct electron paramagnetic resonance (EPR) in the presence and absence of different liposomes. Direct low-temperature (11 degrees K) EPR analysis of the cytochrome c heme iron on exposure to tert-BuOOH shows a gradual (180 s) conversion of the low-spin form to a high-spin Fe(III) species of rhombic symmetry (g = 4.3), with disappearance of a prior peroxyl radical signal (g(o) = 2.014). The conversion to high spin precedes Soret band bleaching, observable by UV/Vis spectroscopy and by magnetic circular dichroism (MCD) at room temperature, that indicates loss of iron coordination by the porphyrin ring. The presence of cardiolipin-containing liposomes delayed formation of the peroxyl radical and conversion to high-spin iron, while dicetylphosphate (DCP) liposomes accelerated these changes. Correspondingly, bleaching of cytochrome c by tert-BuOOH at room temperature was accelerated by several negatively charged liposome preparations, and inhibited by mitochondrial-mimetic phosphatidylcholinephosphatidylethanolaminecardiolipin (PCPECL) liposomes. Concomitant with bleaching, spin-trapping measurements with 5,5-dimethyl-1-pyroline-N-oxide showed that while the relative production of peroxyl, alkoxyl, and alkyl radicals was unaffected by DCP liposomes, PCPECL liposomes decreased the spin-trapped alkoxyl radical signal by 50%. The EPR results show that the primary initial change on exposure of cytochrome c to tert-BuOOH is a change to a high-spin Fe(III) species, and together with MCD measurements show that unsaturated cardiolipin-containing lipid membranes influence the interaction of tert-BuOOH with cytochrome c heme iron, to alter radical production and decrease damage to the cytochrome.     

Redox properties of human endothelial nitric-oxide synthase oxygenase and reductase domains purified from yeast expression system

Characterization of the redox properties of endothelial nitric-oxide synthase (eNOS) is fundamental to understanding the complicated reaction mechanism of this important enzyme participating in cardiovascular function. Yeast overexpression of both the oxygenase and reductase domains of human eNOS, i.e. eNOS(ox) and eNOS(red), has been established to accomplish this goal. UV-visible and electron paramagnetic resonance (EPR) spectral characterization for the resting eNOS(ox) and its complexes with various ligands indicated a standard NOS heme structure as a thiolate hemeprotein. Two low spin imidazole heme complexes but not the isolated eNOS(ox) were resolved by EPR indicating slight difference in heme geometry of the dimeric eNOS(ox) domain. Stoichiometric titration of eNOS(ox) demonstrated that the heme has a capacity for a reducing equivalent of 1-1.5. Additional 1.5-2.5 reducing equivalents were consumed before heme reduction occurred indicating the presence of other unknown high potential redox centers. There is no indication for additional metal centers that could explain this extra electron capacity of eNOS(ox). Ferrous eNOS(ox), in the presence of l-arginine, is fully functional in forming the tetrahydrobiopterin radical upon mixing with oxygen as demonstrated by rapid-freeze EPR measurements. Calmodulin binds eNOS(red) at 1:1 stoichiometry and high affinity. Stoichiometric titration and computer simulation enabled the determination for three redox potential separations between the four half-reactions of FMN and FAD. The extinction coefficient could also be resolved for each flavin for its semiquinone, oxidized, and reduced forms at multiple wavelengths. This first redox characterization on both eNOS domains by stoichiometric titration and the generation of a high quality EPR spectrum for the BH(4) radical intermediate illustrated the usefulness of these tools in future detailed investigations into the reaction mechanism of eNOS.