Direct detection of a dioxygen adduct of cytochrome a3 in the mixed valence cytochrome oxidase/dioxygen reaction

Time-resolved resonance Raman spectra have been recorded during the reaction of mixed valence (a3+ a2+(3)) cytochrome oxidase with dioxygen at room temperature. In the spectrum recorded at 10 microseconds subsequent to carbon monoxide photolysis, a mode is observed at 572 cm-1 that shifts to 548 cm-1 when the experiment is repeated with 18O2. The appearance of this mode is dependent upon the laser intensity used and disappears at higher incident energies. The high frequency data in conjunction with the mid-frequency data allow us to assign the 572 cm-1 mode to the Fe-O stretching vibration of the low-spin O2 adduct that forms in the mixed valence cytochrome oxidase/dioxygen reaction. The 572 cm-1 v(Fe2(+)-O2) frequency in the mixed valence enzyme/O2 adduct is essentially identical to the 571 cm-1 frequency we measured for this mode during the reduction of O2 by the fully reduced enzyme (Varotsis, C., Woodruff, W. H., and Babcock, G. T. (1989) J. Am. Chem. Soc. 111, 6439-6440; Varotsis, C., Woodruff, W. H., and Babcock, G. T. (1990) J. Am. Chem. Soc. 112, 1297), which indicates that the O2-bound cytochrome a3 site is independent of the redox state of the cytochrome a/CuA pair. The photolabile oxy intermediate is replaced by photostable low- or intermediate-spin cytochrome a3+(3), with t1/2 congruent to 200 microseconds.     

Direct detection of Fe(IV)[double bond]O intermediates in the cytochrome aa3 oxidase from Paracoccus denitrificans/H2O2 reaction

We report the first evidence for the formation of the "607- and 580-nm forms" in the cytochrome oxidase aa3/H2O2 reaction without the involvement of tyrosine 280. The pKa of the 607-580-nm transition is 7.5. The 607-nm form is also formed in the mixed valence cytochrome oxidase/O2 reaction in the absence of tyrosine 280. Steady-state resonance Raman characterization of the reaction products of both the wild-type and Y280H cytochrome aa3 from Paracoccus denitrificans indicate the formation of six-coordinate low spin species, and do not support, in contrast to previous reports, the formation of a porphyrin pi-cation radical. We observe three oxygen isotope-sensitive Raman bands in the oxidized wild-type aa3/H2O2 reaction at 804, 790, and 358 cm-1. The former two are assigned to the Fe(IV)[double bond]O stretching mode of the 607- and 580-nm forms, respectively. The 14 cm-1 frequency difference between the oxoferryl species is attributed to variations in the basicity of the proximal to heme a3 His-411, induced by the oxoferryl conformations of the heme a3-CuB pocket during the 607-580-nm transition. We suggest that the 804-790 cm-1 oxoferryl transition triggers distal conformational changes that are subsequently communicated to the proximal His-411 heme a3 site. The 358 cm-1 mode has been found for the first time to accumulate with the 804 cm-1 mode in the peroxide reaction. These results indicate that the mechanism of oxygen reduction must be reexamined.     

Primary intermediate in the reaction of mixed-valence cytochrome c oxidase with oxygen

The reaction of dioxygen with mixed-valence cytochrome c oxidase was followed in a rapid-mixing continuous-flow apparatus. The optical absorption difference spectrum and a kinetic analysis confirm the presence of the primary oxygen intermediate in the 0-100-microseconds time window. The resonance Raman spectrum of the iron-dioxygen stretching mode (568 cm-1) supplies evidence that the degree of electron transfer from the iron atom to the dioxygen is similar to that in oxy complexes of other heme proteins. Thus, the Fe-O2 bond does not display any unique structural features that could account for the rapid reduction of dioxygen to water. Furthermore, the frequency of the iron-dioxygen stretching mode is the same as that of the primary intermediate in the fully reduced enzyme, indicating that the oxidation state of cytochrome a plays no role in controlling the initial properties of the oxygen binding site.     

Resonance Raman study on photoreduction of cytochrome c oxidase: distinction of cytochromes a and a3 in the intermediate oxidation states

Occurrence of photoreduction of bovine cytochrome c oxidase was confirmed with the difference absorption spectra and oxygen consumption measurements for the enzyme irradiated with laser light at 406.7, 441.6, and 590 nm. The resonance Raman spectra were obtained under the same experimental conditions as those adopted for the measurements of oxygen consumption and difference absorption spectra. The photoreduction was more effective upon irradiation at shorter wavelengths and was irreversible under anaerobic conditions. However, upon aeration into the cell, the original oxidized form was restored. It was found that aerobic laser irradiation produces a photo steady state of the catalytic dioxygen reduction and that the Raman scattering from this photo steady state probes cytochrome a2+ and cytochrome a3(3)+ separately upon excitations at 441.6 and 406.7 nm, respectively. The enzyme was apparently protected from the photoreduction in the spinning cell with the spinning speed between 1 and 1500 rpm. These results were explained satisfactorily with the reported rate constant for the electron transfer from cytochrome a to cytochrome a3 (0.58 s-1) and a comparable photoreduction rate of cytochrome a. The anaerobic photoreduction did give Raman lines at 1666 and 214 cm-1, which are characteristic of the ferrous high-spin cytochrome a3(2)+, but they were absent under aerobic photoreduction. The formyl CH = O stretching mode of the a3 heme was observed at 1671 cm-1 for a2+a3(2)+CO but at 1664 cm-1 for a2+a3(2)+CN-, indicating that the CH = O stretching frequency reflects the pi back-donation to the axial ligand similar to the oxidation state marker line (v4).     

Optical and resonance Raman spectroscopy of the heme groups of the quinol-oxidizing cytochrome aa3 of Bacillus subtilis.

The cytochrome aa3-type terminal quinol oxidase of Bacillus subtilis catalyzes the four-electron reduction of dioxygen to water. It resembles the aa3-type cytochrome-c oxidase in using heme A as its active-site chromophores but lacks the CuA center and the cytochrome-c oxidizing activity of the mitochondrial enzyme. We have used optical and resonance Raman spectroscopies to study the B. subtilis oxidase in detail. The alpha-band absorption maximum of the reduced minus oxidized enzyme is shifted by 5-7 nm to the blue relative to most other aa3-type oxidases, and accordingly, we designate the Bacillus enzyme as cytochrome aa3-600. The shifted optical spectrum cannot be ascribed to an alteration in the strength of the hydrogen bond between the formyl group of the low-spin heme and its environment, as the Raman line assigned to this mode in aa3-600 has the same frequency and degree of resonance enhancement as the low-spin heme a formyl mode in most other aa3-type oxidases. Raman modes arise at 194 and 214 cm-1 in aa3-600, whereas a single band at about 214 cm-1 is assigned to the iron-histidine stretch for the other aa3-type oxidases. Possible explanations for the occurrence of these two modes are discussed. Comparison of formyl and vinyl modes and heme skeletal vibrational modes in different oxidation states of aa3-600 and of beef heart cytochrome-c oxidase shows a strong similarity, which suggests conservation of essential features of the heme environments in these oxidases.     

Identification of a ferryl intermediate of Escherichia coli cytochrome d terminal oxidase by resonance Raman spectroscopy.

The 680-nm-absorbing "peroxide state" of the Escherichia coli cytochrome d terminal oxidase complex, obtained by addition of excess hydrogen peroxide to the enzyme, is shown to be a ferryl intermediate in the catalytic cycle of the enzyme. This ferryl intermediate is also created by aerobic oxidation of the fully reduced enzyme. Resonance Raman spectra with 647.1-nm excitation show an FeIV = O stretching band at 815 cm-1, a higher frequency than noted in any other ferryl-containing enzyme to date. The band shows an 16O/18O frequency shift of -46 cm-1, larger than that observed for any porphyrin ferryl species. The FeIV = O formulation was unambiguously established by oxidations of the reduced enzyme with 16O2, 18O2, and 16O18O. Only the use of a mixed-isotope gas permitted discrimination between a ferryl and a peroxo structure. A catalytic cycle for the cytochrome d terminal oxidase complex is proposed, and possible reasons for the high v(Fe = O) frequency are discussed.     

Photodissociated cytochrome c oxidase: cryotrapped metastable intermediates

By freezing CO-bound cytochrome c oxidase at cryogenic temperatures, we have been able to cryotrap metastable intermediates of photodissociation. The differences in the resonance Raman spectrum between these intermediates and ligand-free reduced cytochrome oxidase at cryogenic temperatures are the same as those between the phototransient and the fully reduced preparation detected with 10-ns excitation at room temperature. The largest difference occurs in the iron-histidine stretching mode of cytochrome a3, which shifts by up to 8 cm-1 to higher frequency in the photoproduct. At 4 K the iron-histidine mode displays two unrelaxed frequencies in the photoproduct, which we attribute to two different unrelaxed structures of the heme pocket. The frequencies and intensities of the lines in the resonance Raman spectrum are sensitive to the incident laser power density in both the ligand-free fully reduced preparation and the photoproduct even at 4 K. At 77 K the carbonyl stretching mode of the formyl group in cytochrome a32+ is especially sensitive to laser power, displaying two frequencies-1666 cm-1 at low-flux density and 1674 cm-1 at high-flux density. These frequencies may reflect a change in conformation of the formyl group or a change in its interaction with the protein such as in hydrogen bonding to the carbonyl of the formyl group. The absence of immediate relaxation of the CO photoproduct must be considered when one studies the structure and kinetics of the O2 intermediates that are formed in triple trapping and flow-flash experiments following photodissociation of the CO-bound enzyme.     

Nitrosyl cytochrome c oxidase. Formation and properties of mixed valence enzyme

We report the first resonance Raman scattering studies of NO-bound cytochrome c oxidase. Resonance Raman scattering and optical absorption spectra have been obtained on the fully reduced enzyme (a2+, a2+(3) NO) and the mixed valence enzyme (a3+, a2+(3) NO). Clear vibrational frequency shifts are detected in the lines associated with cytochrome a in comparing the two redox states. With 441.6 nm excitation the fully reduced preparation yields a spectrum similar to that of carbon monoxide-bound cytochrome c oxidase and is dominated by the spectrum of reduced cytochrome a. In contrast, in the mixed valence preparation no contributions from reduced cytochrome a are evident in the spectrum, verifying that this heme is no longer in the Fe2+ state. In the mixed valence NO-bound samples, a line appears at approximately 545 cm-1, a frequency similar to that found in NO-bound hemoglobin and myoglobin and assigned as an Fe-N-O-bending mode in those proteins. We do not detect this line in the spectrum of the fully reduced NO-bound enzyme. The carbonyl line of the cytochrome a3 heme formyl group in the fully reduced NO-bound enzyme appears at approximately equal to 1666 cm-1 in the resonance Raman spectrum. In the mixed valence NO-bound preparation the frequency of the carbonyl line increases by 1.2 cm-1 to approximately equal to 1667 cm-1. Thus, modes in cytochrome a2+(3) NO are sensitive to the redox state of the cytochrome a and/or CuA centers. We propose that the redox sensitivity of the formyl mode and the Fe-N-O mode results from an interaction between cytochrome a2+(3) (NO) and the cytochrome a-CuA pair, and is linked to the cytochrome a3 (NO) by the coupling between CuB and the NO-bound cytochrome a3 heme.     

Resonance Raman study of the aa3-type cytochrome oxidase of thermophilic bacterium PS3

Resonance Raman spectra of the aa3-type cytochrome oxidase of thermophilic bacterium PS3, which has a simpler subunit composition than the mitochondrial enzymes but very similar enzymatic properties, are investigated under various conditions and compared with those of mitochondrial enzymes. The intensities of the two marker lines of reduced cytochrome a3 at 1667 and 213 cm-1 had different dependences on the incubation temperatures and pH. With regard to the incubation temperature dependence, the intensity of the 1667-cm-1 line, the peripheral CH = O stretching mode of the a3 heme, behaved in nearly the same way as that of the oxidase activity whereas the intensity of the 213-cm-1 line, the Fe-histidine stretching mode of the a3 heme, exhibited a similar dependence to that of the proton pumping activity. The 213-cm-1 line disappeared upon binding of carbon monoxide, upon raising the pH above 9.2, or after incubating above 55 degrees C. The Raman line at 1611 cm-1, which was recently suggested to probe the proton pump activity [Babcock, G.T., & Callahan, P.M. (1983) Biochemistry 22, 2314-2319], remained unaltered after incubation at 60 degrees C for 20 min despite a reduction of proton pumping activity to one-third. This argues against the proposed mechanism. The frequencies of the Raman lines were the same for the intact membrane and the isolated enzyme in the reduced state. The Raman spectra of cytochrome oxidase isolated from bacterium, yeast, and bovine heart were different in the lower frequency region below 600 cm-1 but closely alike in the higher frequency region above 1200 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new approach for studying fast biological reactions involving dioxygen: the reaction of fully reduced cytochrome c oxidase with O2

We describe a new method for studying rapid biological reactions involving dioxygen. This approach is based on the photolysis of a synthetic caged dioxygen carrier, which produces dioxygen on a fast time scale. The method was used to investigate the reduction of dioxygen to water by cytochrome c oxidase at room temperature following photolysis of a (mu-peroxo)(mu-hydroxo)bis[bis(bipyridyl)c obalt(III)] complex. The fact that dioxygen is generated in situ on a nanosecond or faster time scale avoids potential complications related to the fate of photodissociated CO in a conventional CO flow-flash experiment. The cobalt complex is stable at room temperature under anaerobic conditions and releases dioxygen upon irradiation at 355 nm with a quantum yield of 0.04. The complex does not react with reduced cytochrome oxidase or its reducing agents within the mixing time of the experiment, and its photoproducts do not interfere with the kinetics of the dioxygen reduction. The oxidation of the reduced cytochrome oxidase was monitored between 500 and 750 nm using a gated optical spectrometric multichannel analyzer following photodissociation of the cobalt complex. The data were analyzed using singular value decomposition and global exponential fitting, and two apparent lifetimes (380 +/- 50 micros and 1.7 +/- 0.2 ms) were resolved and compared to results from a conventional CO flow-flash experiment. The results show that approximately 90 microM dioxygen can be generated upon a single laser pulse and that this approach can be used to study other fast biological reactions involving O(2).     

The site and mechanism of dioxygen reduction in bovine heart cytochrome c oxidase

The site and mechanism of dioxygen reduction in cytochrome c oxidase from bovine heart muscle have been investigated. The rate of cytochrome c2+ oxidation by O2 is shown to be affected by several factors: 1) pH, with optima at 5.65 and 6.0, 2) temperature between 0 and 29 degrees C, with E alpha = 13 kcal mol-1, 3) D2O exchange, with a reduction in rate of 50% or more at the pH optima, and 4) the addition of ethylene glycol or glycerol, which significantly lowers the rate. The extremely narrow (delta vCO approximately 4 cm-1) infrared stretch bands at approximately 1964 and approximately 1959 cm-1 for liganded CO are only slightly affected by factors 1-4 or by changes in the oxidation state of metals other than the heme alpha 3 iron. These results indicate a stable, unusually immobile O2 reduction site well-isolated from the external medium, a characteristic expected to be important for oxidase function. Precise stereochemical positioning of hydrogen donors adjacent to O2 liganded to heme alpha 3 iron can be expected in order to achieve the optimization of the time/distance relationships required for enzyme catalysis. These findings support a novel mechanism of O2 reduction via a hydroperoxide intermediate within a reaction pocket that experiences little change in conformation during the hydrogen and electron transfer steps.     

The structure of the paramagnetic oxygen intermediate in the cytochrome c oxidase reaction.

A paramagnetic intermediate with an unusual e.p.r. spectrum is formed when fully reduced cytochrome c oxidase is allowed to react with dioxygen at 173 K. The effect on the e.p.r. spectrum of using dioxygen enriched in 17O was investigated. These experiments show that an oxygen atom derived from dioxygen is bound to Cu2+ in the intermediate. The e.p.r. parameters can be explained in terms of a weak antiferromagnetic interaction (J approximately equal to 10 cm-1) between Cu2+B and cytochrome a3 in the low-spin ferryl ion state. It is suggested that an OH- ion bound to Cu2+B is hydrogen bonded to the oxygen atom of the ferryl ion in cytochrome a3.     

Observation of the Fe4+ = O stretching Raman band for cytochrome oxidase compound B at ambient temperature

Resonance Raman and visible absorption spectra were simultaneously observed for cytochrome oxidase reaction intermediates at 5 degrees C by using the artificial cardiovascular system (Ogura, T., Yoshikawa, S., and Kitagawa, T. (1989) Biochemistry 28, 8022-8027) and a device for Raman/absorption simultaneous measurements (Ogura, T., and Kitagawa, T. (1988) Rev. Sci. Instrum. 59, 1316-1320). The Fe4+ = O stretching (nu FeO) Raman band was observed at 788 cm-1 for compound B for the first time. This band showed the 16O/18O isotopic frequency shift (delta nu FeO) by 40 cm-1, in agreement with that for horseradish peroxidase compound II (nu FeO = 787 cm-1 and delta nu FeO = 34 cm-1). In the time region when the FeII-O2 stretching band for compound A and the nu FeO band for compound B were coexistent, a Raman band assignable to the Fe3+-O-O-Cu2+ linkage was not recognized.     

Observation of the FeIV=O stretching Raman band for a thiolate-ligated heme protein. Compound I of chloroperoxidase.

The FeIV=O stretching vibration has never been identified for a cysteine-coordinated heme enzyme. In this study, resonance Raman and visible absorption spectra were observed simultaneously for transient species in the catalytic reaction of chloroperoxidase with hydrogen peroxide by using our original apparatus for mixed-flow and Raman/absorption simultaneous measurements. For the first intermediate, the FeIV=O stretching Raman band was observed at 790 cm-1, which shifted to 756 cm-1 with the 18O derivative, but the v4 band was too weak to be identified. This suggested the formation of an oxoferryl porphyrin pi cation radical. The second intermediate gave an intense v4 band at 1,372 cm-1 but no oxygen isotope-sensitive Raman band, suggesting oxygen exchange with bulk water.     

Vibrational structure of the formyl group on heme a. Implications on the properties of cytochrome c oxidase.

Resonance Raman spectra have been recorded for heme a derivatives in which the oxygen atom of the formyl group has been isotopically labeled and for Schiff base derivatives of heme a in which the Schiff base nitrogen has been isotopically labeled. The 14N-15N isotope shift in the C = N stretching mode of the Schiff base is close to the theoretically predicted shift for an isolated C = N group for both the ferric and ferrous oxidation states and in both aqueous and nonaqueous solutions. In contrast, the 16O-18O isotope shift of the C = O stretching mode of the formyl group is significantly smaller than that predicted for an isolated C = O group and is also dependent on whether the environment is aqueous or nonaqueous. This differences between the theoretically predicted shifts and the observed shifts are attributed to coupling of the C = O stretching mode to as yet unidentified modes of the heme. The complex behavior of the C = O stretching vibration precludes the possibility of making simple interpretations of frequency shifts of this mode in cytochrome c oxidase.     

Raman spectra of heme a, cytochrome oxidase-ligand complexes, and alkaline denatured oxidase.

We report 441.6 nm excitation resonance Raman spectra of oxidized and reduced monomeric heme a-imidazole, cytochrome oxidase-exogenous ligand complexes in various redox states, and alkaline denatured oxidase. These data show that, in reduced oxidase, the cytochrome a3 Raman spectrum has bands at 215, 364, 1230, and 1670 cm-1 not observed in the cytochrome a spectrum. The appearance of these bands in the reduced cytochrome a3 spectrum is due to interactions between the heme a of cytochrome a3 and its protein environment and not to intrinsic properties of heme a. These interactions are pH sensitive and strongly influence the vibrational spectra of both heme a groups. We assign the 1670-cm-1 band to the heme a formyl substituent and propose that the intensity of the 1670 cm-1 is high for reduced cytochrome a3 because the C==O lies in the porphyrin plane and is very weak for oxidized and reduced cytochrome a, oxidized cytochrome a3, and oxidized and reduced heme a-imidazole because the C==O lies out of the plane. We suggest that movement of the C==O in and out of the plane explains the ligand induced spectral shift in the optical absorption spectrum of reduced cytochrome a3. Finally, we confirm the observation of Adar & Yonetani (private communication) that, under laser illumination, resting oxidase is photoreactive.     

Resonance Raman evidence for low-spin Fe2+ heme a3 in energized cytochrome c oxidase: implications for the inhibition of O2 reduction

Resonance Raman (RR) spectra are reported for reduced submitochondrial particles (SMP) with excitation at 441.6 nm, where Raman bands of the cytochrome c oxidase heme a groups are selectively enhanced. Addition of ATP to energize the membranes induces the formation of a new band at 1644 cm-1 and partial loss of intensity in a band at 1567 cm-1. These changes are modeled by adding cyanide to reduced cytochrome c oxidase and are attributed to partial conversion of cytochrome (cyt) a3 from a high-spin to a low-spin state. This conversion is abolished by addition of excess oligomycin, an ATPase inhibitor, or FCCP, an uncoupler of proton translocation, and is reversed when the ATP is consumed. The observed spin-state conversion is attributed to the binding of an endogenous ligand to the cyt a3 Fe atom. This ligation is suggested to be induced by a local increase in pH and/or by a global conformation change associated with the generation of a transmembrane potential. Since O2 binding requires a vacant coordination site at cyt a3, the ligation of this site must retard O2 reduction and could thus provide a simple mechanism for energy-linked regulation of respiration. No changes in the RR spectrum were observed upon adding Ca2+ or H+ to reduced cytochrome c oxidase. The cyt a3 spin-state change associated with membrane energization is unrelated to the cyt a absorption red shift induced by adding Ca2+ or H+ to cytochrome c oxidase.     

Resonance Raman spectra of riboflavin and its derivatives in the bound state with egg riboflavin binding proteins

The resonance Raman spectra of riboflavin (RF) and its derivatives, including 3-deuterated (3-D RF), 3-methyl (3-CH3 RF), 3-carboxymethyl (3-CH2COOH RF), and 7,8-dichlororiboflavins (7,8-Cl RF), in H2O and D2O were observed in the 700-1700 cm-1 region. The fluorescence problem of riboflavin was overcome by complex formation of riboflavin with riboflavin binding proteins. The observed frequencies of Raman lines of RF are in good agreement with those of glucose oxidase obtained by Spiro et al. by the resonance CARS method, although the present spectral range is extended to much lower frequency with a higher signal-to-noise ratio than that for glucose oxidase. The observed Raman lines were assigned to the individual ring modes of isoalloxazine on the basis of the Raman spectra of appropriate model compounds such as uracil, pyrazine, and o-xylene. The 1253 cm-1 line of RF was shifted to ca. 1300 cm-1 for 3-D RF, 3-CH3 RF, and 3-CH2COOH RF, and accordingly can be assigned to the CN stretching mode of Ring III. The 1632 cm-1 line of RF was shifted for 7,8-Cl RF and was assigned to a Ring I mode. No Raman line mainly due to C = O stretching mode was observed in the present resonance Raman spectra.     

Kinetic evidence for the re-definition of electron transfer pathways from cytochrome c to O2 within cytochrome oxidase

The reaction with O2 of equimolar mixtures of cytochrome c and cytochrome c oxidase in high and low ionic strength buffers has been examined by flow-flash spectrophotometry at room temperature. In low ionic strength media where cytochrome c and the oxidase are bound in an electrostatic, 1:1 complex some of the cytochrome c is oxidised at a faster rate than a metal centre of the oxidase. In contrast, when cytochrome c and cytochrome c oxidase are predominantly dissociated at high ionic strength cytochrome c oxidation occurs only slowly (t1/2 = 5 s) following the complete oxidation of the oxidase. These results demonstrate that maximal rates of electron transfer from cytochrome c to O2 occur when both substrates are present on the enzyme. The heterogeneous oxidation of cytochrome c observed in the complex implies more than one route for electron transfer within the enzyme. Possibilities for new electron transfer pathways from cytochrome c to O2 are proposed.     

Resonance Raman spectra for catalytic intermediates of cytochrome c oxidase detected with a mixed flow transient apparatus

A novel technique was employed to collect resonance Raman spectra of an oxygenated intermediate of cytochrome c oxidase. Instead of laser pulses of high peak power, which may cause photodissociation, a continuous wave laser and a mixed flow apparatus were used. An intermediate formed within 450 microseconds after the reaction of cytochrome c oxidase with molecular oxygen could be detected. From the spectra it could be deduced that the most likely candidate for the intermediate would be a transient oxygenated species having the Fe2+ - O2 or Fe4+ = O heme in cytochrome a3 and the Fe2+ heme in cytochrome a.