Resonance raman studies of oxo intermediates in the reaction of pulsed cytochrome bo with hydrogen peroxide

Cytochrome bo from Escherichia coli, a member of the heme-copper terminal oxidase superfamily, physiologically catalyzes reduction of O(2) by quinols and simultaneously translocates protons across the cytoplasmic membrane. The reaction of its ferric pulsed form with hydrogen peroxide was investigated with steady-state resonance Raman spectroscopy using a homemade microcirculating system. Three oxygen-isotope-sensitive Raman bands were observed at 805/X, 783/753, and (767)/730 cm(-)(1) for intermediates derived from H(2)(16)O(2)/H(2)(18)O(2). The experiments using H(2)(16)O(18)O yielded no new bands, indicating that all the bands arose from the Fe=O stretching (nu(Fe)(=)(O)) mode. Among them, the intensity of the 805/X cm(-)(1) pair increased at higher pH, and the species giving rise to this band seemed to correspond to the P intermediate of bovine cytochrome c oxidase (CcO) on the basis of the reported fact that the P intermediate of cytochrome bo appeared prior to the formation of the F species at higher pH. For this intermediate, a Raman band assignable to the C-O stretching mode of a tyrosyl radical was deduced at 1489 cm(-)(1) from difference spectra. This suggests that the P intermediate of cytochrome bo contains an Fe(IV)=O heme and a tyrosyl radical like compound I of prostaglandin H synthase. The 783/753 cm(-)(1) pair, which was dominant at neutral pH and close to the nu(Fe)(=)(O) frequency of the oxoferryl intermediate of CcO, presumably arises from the F intermediate. On the contrary, the (767)/730 cm(-)(1) species has no counterpart in CcO. Its presence may support the branched reaction scheme proposed previously for O(2) reduction by cytochrome bo.     

Resonance Raman characterization of the P intermediate in the reaction of bovine cytochrome c oxidase

Reduced cytochrome c oxidase binds molecular oxygen, yielding an oxygenated intermediate first (Oxy) and then converts it to water via the reaction intermediates of P, F, and O in the order of appearance. We have determined the iron-oxygen stretching frequencies for all the intermediates by using time-resolved resonance Raman spectroscopy. The bound dioxygen in Oxy does not form a bridged structure with Cu(B) and the rate of the reaction from Oxy to P (P(R)) is slower at higher pH in the pH range between 6.8 and 8.0. It was established that the P intermediate has an oxo-heme and definitely not the Fe(a(3))-O-O-Cu(B) peroxy bridged structure. The Fe(a(3))=O stretching (nu(Fe=O)) frequency of the P(R) intermediate, 804/764 cm(-1) for (16)O/(18)O, is distinctly higher than that of F intermediate, 785/750 cm(-1). The rate of reaction from P to F in D(2)O solution is evidently slower than that in H(2)O solution, implicating the coupling of the electron transfer with vector proton transfer in this process. The P intermediate (607-nm form) generated in the reaction of oxidized enzyme with H(2)O(2) gave the nu(Fe=O) band at 803/769 cm(-1) for H(2)(16)O(2)/H(2)(18)O(2) and the simultaneously measured absorption spectrum exhibited the difference peak at 607 nm. Reaction of the mixed valence CO adduct with O(2) provided the P intermediate (P(M)) giving rise to an absorption peak at 607 nm and the nu(Fe=O) bands at 804/768 cm(-1). Thus, three kinds of P intermediates are considered to have the same oxo-heme a(3) structure. The nu(4) and nu(2) modes of heme a(3) of the P intermediate were identified at 1377 and 1591 cm(-1), respectively. The Raman excitation profiles of the nu(Fe=O) bands were different between P and F. These observations may mean the formation of a pi cation radical of porphyrin macrocycle in P.     

Resonance Raman applications in investigations of cytochrome c oxidase

Recent applications of resonance Raman (RR) spectroscopy in investigations of cytochrome c oxidase (CcO) are reviewed. Red-excited RR spectra for the fully oxidized "as-isolated" CcO tuned to the ligand-to-metal charge transfer absorption band at 655nm exhibit a Raman band at 755cm(-1) assignable to the ν(OO) stretching mode of a peroxide. Binding of CN(-) diminishes the RR band concomitant with the loss of the charge transfer absorption band. This suggests that a peroxide forms a bridge between heme a(3) and Cu(B). Time-resolved RR spectroscopy of whole mitochondria identified a band at 571cm(-1) arising from the oxygenated intermediate at Δt=0.4, 0.6 and 1.4ms. Bands at 804 and 780cm(-1) of the P and F intermediates were observed at Δt=0.6 and 1.4ms, respectively. The coordination geometries of the three intermediates are essentially the same as the respective species observed for solubilized CcO. However, the lifetime of the oxygenated intermediate in mitochondria was significantly longer than the lifetime of this intermediate determined for solubilized CcO. This phenomenon is due either to the pH effect of mitochondrial matrix, the effect of ΔpH and/or ΔΨ across the membrane, or the effect of interactions with other membrane components and/or phospholipids.     

Implications of ligand binding studies for the catalytic mechanism of cytochrome c oxidase

The reaction of oxidized bovine heart cytochrome c oxidase (CcO) with one equivalent of hydrogen peroxide results in the formation of two spectrally distinct species. The yield of these two forms is controlled by the ionization of a group with a pK(a) of 6.6. At basic pH, where this group is deprotonated, an intermediate called P dominates (P, because it was initially believed to be a peroxy compound). At acidic pH where the group is protonated, a different species, called F (ferryl intermediate) is obtained. We previously proposed that the only difference between these two species is the presence of one proton in the catalytic center of F that is absent in P. It is now suggested that the catalytic center of this F form has the same redox and protonation state as a second ferryl intermediate produced at basic pH by two equivalents of hydrogen peroxide; the role of the second equivalent of H(2)O(2) is that of a proton donor in the conversion of P to F. Two chloride-binding sites have been detected in oxidized CcO. One site is located at the binuclear center; the second site was identified from the sensitivity of g=3 signal of cytochrome a to chloride in the EPR spectra of oxidized CcO. Turnover of CcO releases chloride from the catalytic center into the medium probably by one of the hydrophobic channels, proposed for oxygen access, with an orientation parallel to the membrane plane. Chloride in the binuclear center is most likely not involved in CcO catalysis. The influence of the second chloride site upon several reactions of CcO has been assessed. No correlation was found between chloride binding to the second site and the reactions that were examined.     

Theoretical study on electronic structures of FeOO, FeOOH, FeO(H2O), and FeO in hemes: as intermediate models of dioxygen reduction in cytochrome c oxidase

The electronic structures of heme-dioxygen complexes have been studied as intermediate models of dioxygen reduction mechanism catalyzed by the mixed valence (MV) and fully reduced (FR) cytochrome c oxidase (CcO). Dioxygen, protons and electrons were sequentially added to the heme along the proposed reaction path for the O(2) reduction mechanism. The electronic structures of [FeOO], [FeOO](-), [FeOOH](+), [FeOOH], [Fe=O, H(2)O](+), [Fe=O](+) and [Fe=O] were thoroughly investigated by using the unrestricted hybrid exchange-correlation functional B3LYP method. The additions of two protons and an electron to [FeOO] lead to the OO bond cleavage to produce a H(2)O molecule with the electron transfer from Fe(II) in heme to the OO moiety. It is shown that the intrinsic OO bond cleavage occurs by adding two protons and two electrons into the OO bond, indicating consistency with a H(2)O formation catalyzed by both MV and FR CcO. The study of the electronic structures of heme-dioxygen complexes gave the different proposals for the mechanisms of a H(2)O formation by both MV and FR CcO. For the mixed valence CcO, starting from the [FeOO] complex, the final products are single H(2)O molecule and compound I of the oxo heme. For the fully reduced CcO, the final products are single H(2)O molecule and compound II of the oxo heme which is a reduced state of the compound I.     

The dioxygen cycle. Spectral, kinetic, and thermodynamic characteristics of ferryl and peroxy intermediates observed by reversal of the cytochrome oxidase reaction.

The catalytic mechanism of O2 reduction by cytochrome oxidase was studied in isolated mitochondria and mitoplasts by partial reversal of the reaction. At a high redox potential (Eh) of cytochrome c, high pH, and a high electrochemical proton gradient (delta mu H+) across the inner mitochondrial membrane, the initial ferriccupric state (O) of the oxidized enzyme's bimetallic oxygen reaction center is converted to ferryl (F) and peroxy (P) intermediates, the optical spectroscopic properties of which are reported in detail. This is associated with reversed electron transfer from the bimetallic center to ferricytochrome c. The kinetics of reduction of ferricytochrome c by the reversed electron transfer process are compared with the kinetics of formation of F and P. The results are consistent with transfer of one electron from the ferric-cupric bimetallic center (O) to cytochrome c, yielding the F intermediate, followed by transfer of one electron from the latter to cytochrome c, yielding the P state. In the absence of an effective redox buffer, poising cytochrome c highly oxidized, these primary events are immediately followed by reoxidation of cytochrome c, which is ascribed to forward electron transfer to enzyme molecules still in the O state. This forward reaction also results in accumulation of the P intermediate. Kinetic stimulations of the data predict equilibrium constants for the reversed electron transfer steps, and Em,7 values of approximately 1.1 and 1.2 V may be calculated for the F/O and P/F redox couples, respectively, at delta mu H+ and delta psi equal to zero. Taken together with previously measured Em,7 values, these data indicate that it is the two-electron reduction of bound dioxygen to bound peroxide that is responsible for the irreversibility of the catalytic dioxygen cycle of cell respiration.     

Observation of a novel transient ferryl complex with reduced CuB in cytochrome c oxidase

The reaction between mixed-valence (MV) cytochrome c oxidase from beef heart with H2O2 was investigated using the flow-flash technique with a high concentration of H2O2 (1 M) to ensure a fast bimolecular interaction with the enzyme. Under anaerobic conditions the reaction exhibits 3 apparent phases. The first phase (tau congruent with 25 micros) results from the binding of one molecule of H2O2 to reduced heme a3 and the formation of an intermediate which is heme a3 oxoferryl (Fe4+=O2-) with reduced CuB (plus water). During the second phase (tau congruent with 90 micros), the electron transfer from CuB+ to the heme oxoferryl takes place, yielding the oxidized form of cytochrome oxidase (heme a3 Fe3+ and CuB2+, plus hydroxide). During the third phase (tau congruent with 4 ms), an additional molecule of H2O2 binds to the oxidized form of the enzyme and forms compound P, similar to the product observed upon the reaction of the mixed-valence (i.e., two-electron reduced) form of the enzyme with dioxygen. Thus, within about 30 ms the reaction of the mixed-valence form of the enzyme with H2O2 yields the same compound P as does the reaction with dioxygen, as indicated by the final absorbance at 436 nm, which is the same in both cases. This experimental approach allows the investigation of the form of cytochrome c oxidase which has the heme a3 oxoferryl intermediate but with reduced CuB. This state of the enzyme cannot be obtained from the reaction with dioxygen and is potentially useful to address questions concerning the role of the redox state in CuB in the proton pumping mechanism.     

A combined quantum chemical and crystallographic study on the oxidized binuclear center of cytochrome c oxidase

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain. By reducing oxygen to water, it generates a proton gradient across the mitochondrial or bacterial membrane. Recently, two independent X-ray crystallographic studies ((Aoyama et al. Proc. Natl. Acad. Sci. USA 106 (2009) 2165-2169) and (Koepke et al. Biochim. Biophys. Acta 1787 (2009) 635-645)), suggested that a peroxide dianion might be bound to the active site of oxidized CcO. We have investigated this hypothesis by combining quantum chemical calculations with a re-refinement of the X-ray crystallographic data and optical spectroscopic measurements. Our data suggest that dianionic peroxide, superoxide, and dioxygen all form a similar superoxide species when inserted into a fully oxidized ferric/cupric binuclear site (BNC). We argue that stable peroxides are unlikely to be confined within the oxidized BNC since that would be expected to lead to bond splitting and formation of the catalytic P intermediate. Somewhat surprisingly, we find that binding of dioxygen to the oxidized binuclear site is weakly exergonic, and hence, the observed structure might have resulted from dioxygen itself or from superoxide generated from O(2) by the X-ray beam. We show that the presence of O(2) is consistent with the X-ray data. We also discuss how other structures, such as a mixture of the aqueous species (H(2)O+OH(-) and H(2)O) and chloride fit the experimental data.     

Involvement of phospholipid, biomembrane integrity, and NO peroxidase activity in the NO catabolism by cytochrome c oxidase

The physiological regulation of mitochondrial respiration by NO has been reported to result from the reversible binding of NO to the two-electron reduced binuclear center (Fe(2+)(a3)-Cu(1+)(B)) of cytochrome c oxidase (CcO). Although the role of CcO and its derived catalytic intermediates in the catabolism of NO has been documented, little has been established for the enzyme in its fully oxidized state (Fe(3+)(a3)-Cu(2+)(B)). We report: (1) CcO, in its fully oxidized state, represents the major component of the mitochondrial electron transport chain for NO consumption as controlled by the binding of NO to its binuclear center. Phospholipid enhances NO consumption by fully oxidized CcO, whereas the consumption of NO is slowed down by membrane structure and membrane potential when CcO is embedded in the phospholipid bilayer. (2) In the presence of H(2)O(2), CcO was shown to serve as a mitochondria-derived NO peroxidase. A CcO-derived protein radical intermediate was induced and involved in the modulation of NO catabolism.     

Active site structure of SoxB-type cytochrome bo3 oxidase from thermophilic Bacillus

Two-subunit SoxB-type cytochrome c oxidase in Bacillus stearothermophilus was over-produced, purified, and examined for its active site structures by electron paramagnetic resonance (EPR) and resonance Raman (RR) spectroscopies. This is cytochrome bo3 oxidase containing heme B at the low-spin heme site and heme O at the high-spin heme site of the binuclear center. EPR spectra of the enzyme in the oxidized form indicated that structures of the high-spin heme O and the low-spin heme B were similar to those of SoxM-type oxidases based on the signals at g=6.1, and g=3.04. However, the EPR signals from the CuA center and the integer spin system at the binuclear center showed slight differences. RR spectra of the oxidized form showed that heme O was in a 6-coordinated high-spin (nu3 = 1472 cm(-1)), and heme B was in a 6-coordinated low-spin (nu3 = 1500 cm(-1)) state. The Fe2+-His stretching mode was observed at 211 cm(-1), indicating that the Fe2+-His bond strength is not so much different from those of SoxM-type oxidases. On the contrary, both the Fe2+-CO stretching and Fe2+-C-O bending modes differed distinctly from those of SoxM-type enzymes, suggesting some differences in the coordination geometry and the protein structure in the proximity of bound CO in cytochrome bo3 from those of SoxM-type enzymes.     

Raman evidence for specific substrate-induced structural changes in the heme pocket of human cytochrome P450 aromatase during the three consecutive oxygen activation steps

Specific substrate-induced structural changes in the heme pocket are proposed for human cytochrome P450 aromatase (P450arom) which undergoes three consecutive oxygen activation steps. We have experimentally investigated this heme environment by resonance Raman spectra of both substrate-free and substrate-bound forms of the purified enzyme. The Fe-CO stretching mode (nu(Fe)(-)(CO)) of the CO complex and Fe(3+)-S stretching mode (nu(Fe)(-)(S)) of the oxidized form were monitored as a structural marker of the distal and proximal sides of the heme, respectively. The nu(Fe)(-)(CO) mode was upshifted from 477 to 485 and to 490 cm(-)(1) by the binding of androstenedione and 19-aldehyde-androstenedione, substrates for the first and third steps, respectively, whereas nu(Fe)(-)(CO) was not observed for P450arom with 19-hydroxyandrostenedione, a substrate for the second step, indicating that the heme distal site is very flexible and changes its structure depending on the substrate. The 19-aldehyde-androstenedione binding could reduce the electron donation from the axial thiolate, which was evident from the low-frequency shift of nu(Fe)(-)(S) by 5 cm(-)(1) compared to that of androstenedione-bound P450arom. Changes in the environment in the heme distal site and the reduced electron donation from the axial thiolate upon 19-aldehyde-androstenedione binding might stabilize the ferric peroxo species, an active intermediate for the third step, with the suppression of the formation of compound I (Fe(4+)=O porphyrin(+)(*)) that is the active species for the first and second steps. We, therefore, propose that the substrates can regulate the formation of alternative reaction intermediates by modulating the structure on both the heme distal and proximal sites in P450arom.     

ATR-FTIR spectroscopy of the P(M) and F intermediates of bovine and Paracoccus denitrificans cytochrome c oxidase

The structures of P(M) and F intermediates of bovine and Paracoccus denitrificans cytochrome c oxidase were investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Transitions from the "fast" oxidized state to the P(M) or F states were initiated by perfusion with buffer containing either CO/oxygen or H(2)O(2). Intermediates were quantitated by simultaneous monitoring of visible absorption changes in the protein film. For both bovine and P. denitrificans oxidase, the major features of the IR difference spectrum of P(M) were similar when produced by CO/oxygen or by H(2)O(2) treatments. These IR difference spectra were distinctly different from the IR difference spectrum of F that formed with extended treatment with H(2)O(2). Some IR bands could be assigned tentatively to perturbations of heme a(3) ring modes and substituents, and these perturbations were greater in P(M) than in F. Other bands could be assigned to surrounding protein changes. Strong perturbation of the environment of a carboxylic acid, most likely E-242 (bovine numbering), occurred in P(M) and relaxed back in F. A second redox-sensitive carboxylic acid was also perturbed in the bovine P(M) intermediate. Further consistent signatures of P(M) in both oxidases that were absent in F were strong negative bands at 1547 and 1313 cm(-1) in bovine oxidase (1542 and 1314 cm(-1) in P. denitrificans) and a positive band at approximately 1519 cm(-1). From comparison with available IR data on model compounds, it is suggested that these reflect changes in the covalent tyrosine-histidine ligand to Cu(B). These findings are discussed in relation to the oxidase catalytic cycle.     

Facile electron transfer during formation of cluster X and kinetic competence of X for tyrosyl radical production in protein R2 of ribonucleotide reductase from mouse.

The kinetics and mechanism of formation of the tyrosyl radical and mu-(oxo)diiron(III) cluster in the R2 subunit of ribonucleotide reductase from mouse have been examined by stopped-flow absorption and freeze-quench electron paramagnetic resonance and M?ssbauer spectroscopies. The reaction comprises (1) acquisition of Fe(II) ions by the R2 apo protein, (2) activation of dioxygen at the resulting carboxylate-bridged diiron(II) cluster to form oxidized intermediate diiron species, and (3) univalent oxidation of Y177 by one of these intermediates to form the stable radical, with concomitant or subsequent formation of the adjacent mu-(oxo)diiron(III) cluster. The data establish that an oxidized diiron intermediate spectroscopically similar to the well-characterized, formally Fe(III)Fe(IV) cluster X from the reaction of the Escherichia coli R2 protein precedes the Y177 radical in the reaction sequence and is probably the Y177 oxidant. As formation of the X intermediate (1) requires transfer of an "extra" reducing equivalent to the buried diiron cluster following the addition of dioxygen and (2) is observed to be rapid relative to other steps in the reaction, the present data indicate that the transfer of this reducing equivalent is not rate-limiting for Y177 radical formation, in contrast to what was previously proposed (Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gr?slund, A. (1998) J. Biol. Chem. 273, 21463-21472). Indeed, the formation of X (k(obs) = 13 +/- 3 s(-1) at 5 degrees C and 0.95 mM O(2)) and the decay of the intermediate to give the Y177 radical (k(obs) = 5 +/- 2 s(-1)) are both considerably faster than the formation of the reactive Fe(II)-R2 complex from the apo protein and Fe(II)(aq) (k(obs) = 0.29 +/- 0.03 s(-1)), which is the slowest step overall. The conclusions that cluster X is an intermediate in Y177 radical formation and that transfer of the reducing equivalent is relatively facile imply that the mouse R2 and E. coli R2 reactions are mechanistically similar.     

Heme-copper/dioxygen adduct formation relevant to cytochrome c oxidase: spectroscopic characterization of [(6L)FeIII-(O2 2−)-CuII]+

In the further development and understanding of heme-copper dioxygen reactivity relevant to cytochrome c oxidase O(2)-reduction chemistry, we describe a high-spin, five-coordinate dioxygen (peroxo) adduct of an iron(II)-copper(I) complex, [((6)L)Fe(II)Cu(I)](BArF(20)) (1), where (6)L is a tetraarylporphyrinate with a tethered tris(2-pyridylmethyl)amine chelate for copper. Reaction of 1 with O(2) in MeCN affords a remarkably stable [t(1/2) (rt; MeCN) approximately 60 min] adduct, [((6)L)Fe(III)-(O(2) (2-))-Cu(II)](+) (2) [EPR silent; lambda(max)=418 (Soret), 561 nm], formulated as a peroxo complex based on manometry (1:O(2)=1:1; spectrophotometric titration, -40 degrees C, MeCN), mass spectrometry {MALDI-TOF-MS: (16)O(2), m/z 1191 ([((6)L)Fe(III)-((16)O(2) (2-))-Cu(II)](+)); (18)O(2), m/z 1195}, and resonance Raman spectroscopy (nu((O-O))=788 cm(-1); Delta(16)O(2)/(18)O(2)=44 cm(-1); Delta(16)O(2)/(16/18)O(2)=22 cm(-1)). (1)H and (2)H NMR spectroscopy (-40 degrees C, MeCN) reveals that 2 is the first heme-copper peroxo complex which is high-spin, with downfield-shifted pyrrole resonances (delta(pyrrole)=75 ppm, s, br) and upfield shifted peaks at delta= -22, -35, and -40 ppm, similar to the pattern observed for the mu-oxo complex [((6)L)Fe(III)-O-Cu(II)](BAr(F)) (3) (known S=2 system, antiferromagnetically coupled high-spin Fe(III) and Cu(II)). The corresponding magnetic moment measurement (Evans method, CD(3)CN, -40 degrees C) also confirms the S=2 spin state, with mu(B)=4.9. Structural insights were obtained from X-ray absorption spectroscopy, showing Fe-O (1.83 A) and Cu-O (1.882 A) bonds, and an Fe...Cu distance of 3.35(2) A, suggestive of a mu-1,2-peroxo ligand present in 2. The reaction of 2 with cobaltocene gives 3, differing from the observed full reduction seen with other heme-Cu peroxo complexes. Finally, thermal decomposition of 2 yields 3, with concomitant release of 0.5 mol O(2) per mol 2, as confirmed quantitatively by an alkaline pyrogallol dioxygen scavenging solution.     

Ferryl-oxo heme intermediate in the antimalarial mode of action of artemisinin

Fourier transform infrared (FTIR) and resonance Raman (RR) spectroscopies have been employed to investigate the reductive cleavage of the O-O bond of the endoperoxide moiety of the antimalarial drug artemisinin and its analog trioxane alcohol by hemin dimer. We have recorded FTIR spectra in the nu(O-O) and nu(as)(Fe-O-Fe) regions of artemisinin and of the hemin dimer that show the cleavage of the endoperoxide and that of the hemin dimer, respectively. We observed similar results in the trioxane alcohol/hemin dimer reaction. The RR spectrum of the artemisinin/hemin dimer reaction displays a vibrational mode at 850 cm(-1) that shifts to 818 cm(-1) when the experiment is repeated with (18)O-O(18) endoperoxide enriched trioxane alcohol. The frequency of this vibration and the magnitude of the (18)O-O(18) isotopic shift led us to assign the 850 cm(-1) mode to the Fe(IV) = O stretching vibration of a ferryl-xoxo heme intermediate that occurs in the artemisinin/hemin dimer and trioxane alcohol/hemin reactions. These results provide the first direct characterization of the antimalarial mode of action of artemisinin and its trioxane analog, and suggest that artemisinin appears to react with heme molecules that have been incorporated into hemozoin and subsequently the heme performs cytochrome P450-type chemistry.     

The electronic and vibrational structures of iron-oxo porphyrin with a methoxide or cysteinate axial ligand

Hybrid density functional theory (DFT) calculations for the electronic and vibrational structures of compound I species with a methoxide (MeO-) (1) or cysteinate (CysS-) (2) axial ligand are carried out in order to elucidate the natures of a methoxide-coordinating new type of compound I species (Bull. Chem. Soc. Jpn. 71 (1998) 1343) and cysteinate-coordinating compound I species of chloroperoxidase (CPO-I) and cytochrome P450s (P450-I). DFT computations of 1 and 2 demonstrate that these "anionic" ligands are a spin carrier; 70% (80%) of a spin density resides on the O (S) atom of the axial ligand and 30% (20%) is distributed on the porphyrin ring. These results suggest that for the generation of the compound I species, one electron is removed from the iron centers and the rest of the one electron is supplied from the oxidizable axial ligands instead of the iron centers or the porphyrin ring. Vibrational analyses demonstrate that the Fe=O bond is more strongly activated in 1 compared with 2 with the stretching mode at 849 cm(-1) (878 cm(-1)) for the doublet state1a (2a) and at 814 cm(-1) (875 cm(-1)) in the quartet state 1b (2b). This reverse order of the Fe=O bond strength with respect to the axial donor strength should have relevance to the significantly oxidized character of the CysS- axial ligand. In conjunction with the recent results of the extensive resonance Raman (RR) studies, some interpretations of unsettled RR results for compound I of chloroperoxidase (CPO-I) and a synthetic compound I species [O=FeIV(TMP*+)(alcohol)] (J. Am. Chem. Soc. 113 (1991) 6542) concerning the O=Fe stretching frequencies are discussed.     

The ferrous dioxygen complex of the oxygenase domain of neuronal nitric-oxide synthase

The mechanisms by which nitric-oxide synthases (NOSs) bind and activate oxygen at their P450-type heme active site in order to synthesize nitric oxide from the substrate L-arginine are mostly unknown. To obtain information concerning the structure and properties of the first oxygenated intermediate of the enzymatic cycle, we have used a rapid continuous flow mixer and resonance Raman spectroscopy to generate and identify the ferrous dioxygen complex of the oxygenase domain of nNOS (Fe(2+)O(2) nNOSoxy). We detect a line at 1135 cm(-1) in the resonance Raman spectrum of the intermediate formed from 0.6 to 3.0 ms after the rapid mixing of the ferrous enzyme with oxygen that is shifted to 1068 cm(-1) with (18)O(2). This line is assigned as the O-O stretching mode (nu(O-O)) of the oxygenated complex of nNOSoxy. Rapid mixing experiments performed with nNOSoxy saturated with L-arginine or N(omega)-hydroxy-L-arginine, in the presence or absence of (6R)-5,6, 7,8-tetrahydro-L-biopterin, reveal that the nu(O-O) line is insensitive to the presence of the substrate and the pterin. The optical spectrum of this ferrous dioxygen species, with a Soret band wavelength maximum at 430 nm, confirms the identification of the previously reported oxygenated complexes generated by stopped flow techniques.     

pH dependence of the reduction of dioxygen to water by cytochrome c oxidase. 1. The P(R) state is a pH-dependent mixture of three intermediates,A, P,and F

Recent studies on cytochrome oxidase have indicated that the putative "peroxy" intermediate in the catalytic cycle (P(R)) is a mixture of intermediates, including P and F [Sucheta, A., et al. (1998) Biochemistry 37, 17905-17914], and the bench-made P and F forms appear to have the same redox state (Fe(a3)(4+)=O(2-)), but a different protonation state [Fabian, M., and Palmer, G. (2001) Biochemistry 40, 1867-1874]. To explore the possibility that the putative P(R) state is a pH-dependent mixture of intermediates, we investigated the reduction of dioxygen to water by the fully reduced cytochrome oxidase at pH 6.2, 7.5, and 8.5 in the visible and Soret regions (350-800 nm) using the CO flow-flash technique. Singular value decomposition and global exponential fitting of the time-resolved absorption difference spectra resolved five apparent lifetimes. The fastest three (1.5, 13, and 34 micros) were independent of pH, while the two slowest rates (80-240 micros and 1.1-2.4 ms) decreased by a factor of 2-3 as the pH increased. When the time-resolved spectra were analyzed using a unidirectional sequential model, the spectra of the reduced enzyme and the dioxygen-bound intermediate, compound A, were found to be pH-independent. However, the putative P(R) intermediate was best represented by a pH-dependent mixture of compound A, P, and F. The ferryl form was favored at low pH. The subsequent intermediate is a ferryl with a pH-dependent electron transfer equilibrium between heme a and Cu(A), the reduced heme a being favored at low pH. These results suggest a pH-dependent reaction mechanism of the reduction of dioxygen to water by the fully reduced enzyme that is more complex than previously proposed.     

UV optical absorption by protein radicals in cytochrome c oxidase

The UV properties of key oxygen intermediates of cytochrome c oxidase have been investigated by transient absorption spectroscopy. The temporal behavior of P(m) species upon aerobic incubation with CO or in the reaction with H(2)O(2) is closely concurred by a new optical shift at 290/260 nm. In the acid-induced conversion of P(m) to F(*), it is replaced by another shift at 323/288 nm. The wavelength and intensity of the UV signal observed in F(*) match closely the properties of model Trp? in agreement with results of ENDOR studies on this species. The UV spectrum of Tyr* gives the closest match with the 290/260 nm signal observed in P(m). On the basis of analysis of possible UV chromophores in CcO and similarity to Tyr*, the 290/260 nm signal is proposed to originate from the H(240)-Y(244)* site. Possible effects of local environment on UV properties of this site are discussed.     

Examination of the reaction of fully reduced cytochrome oxidase with hydrogen peroxide by flow-flash spectroscopy

The reaction of cytochrome c oxidase with hydrogen peroxide has been of great value in generating and characterizing oxygenated species of the enzyme that are identical or similar to those formed during turnover of the enzyme with dioxygen. Most previous studies have utilized relatively low peroxide concentrations (millimolar range). In the current work, these studies have been extended to the examination of the kinetics of the single turnover of the fully reduced enzyme using much higher concentrations of peroxide to avoid limitations by the bimolecular reaction. The flow-flash method is used, in which laser photolysis of the CO adduct of the fully reduced enzyme initiates the reaction following rapid mixing of the enzyme with peroxide, and the reaction is monitored by observing the absorbance changes due to the heme components of the enzyme. The following reaction sequence is deduced from the data. (1) The initial product of the reaction appears to be heme a(3) oxoferryl (Fe(4+)=O(2)(-) + H(2)O). Since the conversion of ferrous to ferryl heme a(3) (Fe(2+) to Fe(4+)) is sufficient for this reaction, presumably Cu(B) remains reduced in the product, along with Cu(A) and heme a. (2) The second phase of the reaction is an internal rearrangement of electrons and protons in which the heme a(3) oxoferryl is reduced to ferric hydroxide (Fe(3+)OH(-)). In about 40% of the population, the electron comes from heme a, and in the remaining 60% of the population, Cu(B) is oxidized. This step has a time constant of about 65 micros. (3) The third apparent phase of the reaction includes two parallel reactions. The population of the enzyme with an electron in the binuclear center reacts with a second molecule of peroxide, forming compound F. The population of the enzyme with the two electrons on heme a and Cu(A) must first transfer an electron to the binuclear center, followed by reaction with a second molecule of peroxide, also yielding compound F. In each of these reaction pathways, the reaction time is 100-200 micros, i.e., much faster than the rate of reaction of peroxide with the fully oxidized enzyme. Thus, hydrogen peroxide is an efficient trap for a single electron in the binuclear center. (4) Compound F is then reduced by the final available electron, again from heme a, at the same rate as observed for the reduction of compound F formed during the reaction of the fully reduced oxidase with dioxygen. The product is the fully oxidized enzyme (heme a(3) Fe(3+)OH(-)), which reacts with a third molecule of hydrogen peroxide, forming compound P. The rate of this final reaction step saturates at high concentrations of peroxide (V(max) = 250 s(-)(1), K(m) = 350 mM). The data indicate a reaction mechanism for the steady-state peroxidase activity of the enzyme which, at pH 7.5, proceeds via the single-electron reduction of the binuclear center followed by reaction with peroxide to form compound F directly, without forming compound P. Peroxide is an efficient trap for the one-electron-reduced state of the binuclear center. The results also suggest that the reaction of hydrogen peroxide to the fully oxidized enzyme may be limited by the presence of hydroxide associated with the heme a(3) ferric species. The reaction of hydrogen peroxide with heme a(3) is very substantially accelerated by the availability of an electron on heme a, which is presumably transferred to the binuclear center concomitant with a proton that can convert the hydroxide to water, which is readily displaced.