Iron-histidine stretching Raman line and enzymic activities of bovine and bacterial cytochrome c oxidases

Resonance Raman spectra of the reduced form of cytochrome c oxidase isolated from bovine heart and the thermophilic bacterium PS3 were investigated in relation to their H+-pumping- and cytochrome-c-oxidizing activities, which were varied by incubating the enzyme at raised temperatures or at alkaline pH at room temperature. For both the bovine and PS3 enzymes, the intensity of the iron-histidine stretching Raman line of the ferrous a3 heme (214 cm-1) exhibited an incubation-temperature-dependent change, which fell between the similar curves of the H+-pumping and cytochrome-c-oxidizing activities. The intensities of the formyl CH=O stretching Raman line of the ferrous a3 heme (1665 cm-1) as well as of other lines were insensitive to the heat treatment. The iron-histidine stretching Raman line of both enzymes showed pH-dependent intensity change which was nearly parallel with the pH dependence of cytochrome-c-oxidizing activity. Therefore, deprotonation affecting the 214 cm-1 Raman line is responsible for the decrease of activity. This limited alkaline treatment to the PS3 enzyme was reversible and the recovered enzyme exhibited Raman intensities and enzymic activities similar to the native one. However, the neutralized, bovine enzyme with a similar intensity of the 214 cm-1 line showed increased cytochrome-c-oxidizing activity and null H+-pumping activity.     

Resonance Raman evidence for an exchangeable protein hydrogen associated with the heme a group of cytochrome oxidase

When cytochrome-c oxidase is soaked in D2O, downshifts of the cytochrome a formyl C = O stretching mode are seen in the resonance Raman (RR) spectra (413.1 nm excitation) of both the resting and reduced forms. Other changes observed in the reduced protein RR spectra are consistent with involvement of the cytochrome a formyl group in the deuterium effect. The D2O-induced RR changes are fully developed during 3-5 days incubation, but are incomplete after 1 h. Extraction of the heme a chromophore in deuterated solvents eliminates these changes, implying that the exchangeable proton is on a protein group in the cytochrome a pocket which H-bonds to the heme formyl. The rate of the D2O exchange process is unaffected by enzyme turnover, thus reducing the likelihood that the cytochrome a formyl H-bond is directly involved in the redox-linked mechanism of proton pumping.     

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.     

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.     

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.     

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).     

Appearance of the v(FeIV = O) vibration from a ferryl-oxo intermediate in the cytochrome oxidase/dioxygen reaction

Time-resolved resonance Raman spectra have been recorded during the reaction of fully reduced (a2+a3(2+)) cytochrome oxidase with dioxygen at room temperature. In the spectrum recorded at 800 microseconds subsequent to carbon monoxide photolysis, a mode is observed at 790 cm-1 that shifts to 755 cm-1 when the experiment is repeated with 18O2. The frequency of this vibration and the magnitude of the 18O2 isotopic frequency shift lead us to assign the 790-cm-1 mode to the FeIV = O stretching vibration of a ferryl-oxo cytochrome a3 intermediate that occurs in the reaction of fully reduced cytochrome oxidase with dioxygen. The appearance and vibrational frequency of this mode were not affected when D2O was used as a solvent. This result suggests that the ferryl-oxo intermediate is not hydrogen bonded. We have also recorded Raman spectra in the high-frequency (1000-1700 cm-1) region during the oxidase/O2 reaction that show that the oxidation of cytochrome a2+ is biphasic. The faster phase is complete within 100 microseconds and is followed by a plateau region in which no further oxidation of cytochrome a occurs. The plateau persists to approximately 500 microseconds and is followed by the second phase of oxidation. These results on the kinetics of the redox activity of cytochrome a are consistent with the branched pathway discussed by Hill et al. [Hill, B., Greenwood, C., & Nichols, P. (1986) Biochim. Biophys. Acta 853, 91-113] for the oxidation of reduced cytochrome oxidase by O2 at room temperature.     

Resonance Raman scattering from hemoproteins. Effects of ligands upon the Raman spectra of various C-type cytochromes.

Resonance Raman spectra were measured for various C-type cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome f, and alkylated cytochrome c) and a B-type cytochrome (cytochrome b5) in their reduced and oxidized states. (1) For ferrous alkylated cytochrome c, a Raman line sensitive to the replacement of an axial ligand of the heme iron uas found around 1540 cm=1. This ligand-sensitive Raman line indicated the transition from acidic (1545 cm-1) to alkaline (1533 cm-1) forms with pK 7.9. The pH dependence of the Raman spectrum corresponded well to that of the optical absorption spectra. (2) For ferrous cytochrome f, the ligand-sensitive Raman line was found at the same frequency as cytochrome c (1545 cm-1). Accordingly two axial ligands are likely to be histidine and methionine as in cytochrome c. (3) For ferrous cytochrome c3, the frequency of the ligand-sensitive Raman line was between those of cytochrome c and cytochrome b5. Since two axial ligands of the heme iron in cytochrome c3 might be histidines. However, a combination of histidine and methionine as a possible set of two axial ligands was not completely excluded for one or two of the four hemes. (4) In ferrous cytochrome b5, two weak Raman lines appeared at 1302 and 1338 cm-1 instead of the strongest band at 1313 cm-1 of C-type ferrous cytochromes. This suggests the practical use of these bands for the identification of types of cytochromes. The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical disortion of heme.     

Infrared spectroscopic studies of carbonyl horseradish peroxidases.

Infrared difference spectra, FeIIICO vs. FeIII of horseradish peroxidase isoenzymes A2 and C were recorded from 2000 to 1800 cm-1. Under alkaline conditions, pH 9, both isoenzymes exhibit two CO stretching bands, at 1938 and 1925 cm-1 for A2 and at 1933 and 1929 cm-1 for C. As the pH is lowered the low-frequency band for each isoenzyme decreases in intensity with a concommitant appearance and increase in intensity of a band at 1906 and 1905 cm-1 for the A2 and C isoenzymes, respectively. These changes conform to pK values of 6.7 for the A2 and 8.8 for the C isoenzymes of horseradish peroxidase. The interpretation of the infrared results was simplified by the observation that a linear relationship exists between the redox potential, Em7, for the FeIII/FeII system vs. the infrared CO stretching frequency, vCO, for cytochrome a3, hemoglobin, myoglobin, and cytochrome P-450 cam with substrate. This relationship suggests that the primary force altering vCO in these heme proteins is a variation in electron density at the heme iron and not direct protein interactions with the CO ligand. The horseradish peroxidase infrared bands in the 1930-cm-1 region correlate well with this relationship. The large deviation of the 1905-cm-1 band from the linear relationship and its dependence upon hydrogen ion concentration are consistent with horseradish peroxidase having a single CO binding site which can hold in two geometries, one of which contains an amino acid moiety capable of forming a hydrogen bond to the carbonyl oxygen.     

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.     

Resonance Raman study of cytochrome aa3 from Sulfolobus acidocaldarius.

The single subunit terminal oxidase of Sulfolobus acidocaldarius, cytochrome aa3, was studied by resonance Raman spectroscopy. Results on the fully oxidized, the fully reduced, and the reduced carbon monoxide complex are reported and compared with those of eucaryotic cytochrome oxidase. It is shown that in both redox states the hemes a and a3 are in the six-coordinated low-spin and six-coordinated high-spin configuration, respectively. The resonance Raman spectra reveal far-reaching similarities of this archaebacterial with mammalian or plant enzymes except for the reduced form of heme a. The formyl substituent of this heme appears above 1640 cm-1, ruling out significant hydrogen bonding interactions which is in sharp contrast to beef heart cytochrome oxidase. In addition, frequency upshifts of the marker bands v4 and v2 are noted indicating differences in the electron density distribution within the molecular orbitals of the porphyrin.     

Photoreductive titration of the resonance Raman spectra of cytochrome oxidase in whole mitochondria.

A photoreductive titration of the resonance Raman (RR) spectra of cytochrome c oxidase in whole mitochondria was recorded by exploiting the preferential enhancement of the Raman signals of reduced cytochrome oxidase excited at 441.6 nm. When the sample was cooled to about--10 degrees C, it was possible to slow down the photoreductive effect of the laser and to record RR spectra at various states of reduction. Compared to the earliest recorded scan (most oxidized), the dithionite-reduced sample shows the appearance of new bands at 216, 363, 560, and 1665 cm-1. At intermediate stages of photoreduction, the 216- and 560-cm-1 bands appear before the 363- and 1665-cm-1 bands; photoreduction induces full intensity in the former bands, whereas the latter bands are photoreduced to 50% of the dithionite-reduced intensity. The relative intensities of a doublet at 1609--1623 cm-1 are affected by reduction: the band at 1609 cm-1 is weaker in the earlier scans; in later scans this band has grown to equal intensity with the 1623-cm-1 band. We conclude that this reductive titration of the RR spectrum of cytochrome c oxidase reflects three states in its reduction. The behavior of the doublet at 1609--1623 cm-1 suggests that the two hemes are nonequivalent but interacting. The band at 216 cm-1 may be indicative of an iron-copper interaction that is affected by the presence of external ligands.     

Accessibility of the cytochrome a heme in cytochrome c oxidase to exchangeable protons

The comparison of the resonance Raman spectrum of cytochrome a2+ from cytochrome oxidase in deuterated buffers to that in protonated buffers reveals many lines that have different frequency or intensity. Some of the frequency differences are very large, e.g. on the order of 10 cm-1. From these differences in the Raman spectra, we infer that the heme pocket is readily accessible to protons and that labile groups are either on the heme or interact strongly with it. These data suggest the possibility of direct participation in proton translocation and/or oxygen protonation by the heme of cytochrome a.     

Resonance Raman study of cytochrome b562-o complex, a terminal oxidase of Escherichia coli in its ferric, ferrous, and CO-ligated states

Cytochrome b562-o complex, a terminal oxidase in the respiratory chain of aerobically grown Escherichia coli, has been studied by resonance Raman spectroscopy in its air-oxidized, dithionite-reduced, and reduced and CO-ligated states. In the reduced state, with a 406.7-nm excitation, there appeared 1494 and 1473 cm-1 lines, indicating that low spin and high spin components are included in the cytochrome b562-o complex. For the air-oxidized protein, resonance Raman lines were observed at 1372, 1503, and 1580 cm-1 with a 413.1-nm excitation, indicating that there is a ferric low spin heme. In addition, a weak but appreciable Raman line was observed at 1480 cm-1 assignable to a ferric high spin heme. Accordingly, it was concluded that low spin and high spin components are included in the cytochrome b562-o complex in the reduced and the air-oxidized states. In the CO-ligated state, with a defocused laser beam of 413.1 nm, two Raman bands assignable to the Fe-CO stretching mode have been observed at 489 and 523 cm-1, as a major and a minor component, respectively. When the laser beam was focused upon the sample to cause a photodissociation of CO from the heme moiety, the intensity of the major band at 489 cm-1 was reduced as expected. On the other hand, the minor band at 523 cm-1 remained still obvious. It was suggested that the cytochrome b562-o complex may have an additional anomalous site for CO that is resistant to photodissociation.     

Heme pocket interactions in cytochrome c peroxidase studied by site-directed mutagenesis and resonance Raman spectroscopy

Resonance Raman spectra are reported for FeII and FeIII forms of cytochrome c peroxidase (CCP) mutants prepared by site-directed mutagenesis and cloning in Escherichia coli. These include the bacterial "wild type", CCP(MI), and mutations involving groups on the proximal (Asp-235----Asn, Trp-191----Phe) and distal (Trp-51----Phe, Arg-48----Leu and Lys) side of the heme. These spectra are used to assess the spin and ligation states of the heme, via the porphyrin marker band frequencies, especially v3, near 1500 cm-1, and, for the FeII forms, the status of the Fe-proximal histidine bond via its stretching frequency. The FeII-His frequency is elevated to approximately 240 cm-1 in CCP(MI) and in all of the distal mutants, due to hydrogen-bonding interactions between the proximal His-175 N delta and the carboxylate acceptor group on Asp-235. The FeII-His RR band has two components, at 233 and 246 cm-1, which are suggested to arise from populations having H-bonded and deprotonated imidazole; these can be viewed in terms of a double-well potential involving proton transfer coupled to protein conformation. The populations shift with changing pH, possibly reflecting structure changes associated with protonation of key histidine residues, and are influenced by the Leu-48 and Phe-191 mutations. A low-spin FeII form is seen at high pH for the Lys-48, Leu-48, Phe-191, and Phe-51 mutants; for the last three species, coordination of the distal His-52 is suggested by a approximately 200-cm-1 RR band assignable to Fe(imidazole)2 stretching.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome c oxidase: evidence for interaction of water molecules with cytochrome a

The resonance Raman spectra of cytochrome c oxidase in protonated buffer compared to that in deuterated buffer indicate that water molecules are near the heme of cytochrome a. Differences in widths of the heme line at 1610 cm-1, after short exposure to D2O, and, additionally, of the heme line at 1625 cm-1, after long exposure, can be accounted for by changes in resonance vibrational energy transfer between modes of cytochrome a2+ and the bending mode of water molecules in the heme pocket. On the basis of the assignment of these modes, we place one water molecule near the vinyl group and one water molecule near the formyl group of the cytochrome a heme. These water molecules may play several possible functional roles.     

The pH dependence of the resonance raman spectra and structural alterations at heme moieties of various c-type cytochromes.

The pH dependence of resonance Raman spectra were studied for ferrous and ferric cytochromes c, c2, c3, c-551, and c-555. The frequencies of the 1565 cm-1 (ferric) and 1539 cm-1 lines (ferrous) were sensitive to the replacement of the sixth ligand. The titration curve for the 1565 cm-1 line of cytochrome c was parallel with that for the 695 nm band. The pH dependence of the 1539 cm-1 line of ferrous cytochrome c3 suggested the stepwise replacement of the sixth ligand of its four hemes, although such pH dependence was not recognized for the Raman spectra of other ferrous cytochromes investigated. The relative intensities of three Raman lines at 1639, 1587, and 1561 cm-1 of ferric protoporphyrin bis-imidazole complex were changed clearly by the presence of detergents. The relative intensities of the corresponding three Raman lines of cytochromes b5 and c were close to those of the ferric porphyrin complex in the presence and absence of detergents, respectively, suggesting an appreciable difference in their heme environments. Reduced hemin in detergent solution, unexpectedly, gave the Raman spectrum of ferric low spin type.     

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.     

Concerted involvement of cooperative proton-electron linkage and water production in the proton pump of cytochrome c oxidase

In cytochrome c oxidase, oxido-reductions of heme a/Cu(A) and heme a3/Cu(B) are cooperatively linked to proton transfer at acid/base groups in the enzyme. H+/e- cooperative linkage at Fe(a3)/Cu(B) is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H+/e- linkage at heme a (and Cu(A)). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H2O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/Cu(A) and heme a3/Cu(B) in the soluble oxidase; (iii) H+ release in the external phase (i.e. H+ pumping) associated with the oxidative (R-->O transition), reductive (O-->R transition) and a full catalytic cycle (R-->O-->R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H+/e- linkage at heme a/Cu(A) and heme a3/Cu(B) with acid/base clusters, C1 and C2 respectively, and protonmotive steps of the reduction of O2 to water are involved in proton pumping.     

A comparison of the resonance Raman properties of the fast and slow forms of cytochrome oxidase

Resonance Raman (RR) spectra of the "rapid" and "slow" forms (Baker et al., 1987) of resting cytochrome oxidase obtained with Soret excitation at 413.1 nm are reported. There are a number of conspicuous differences between the two forms in the high-frequency region of the RR spectrum which involve changes in Raman intensity arising from a blue shift in the Soret maximum of cytochrome a3 upon conversion to the slow form. In the low-frequency region a peak present at 223 cm-1 in the rapid form shifts to 220 cm-1 in the slow form; this peak is assigned as the cytochrome a3 Fe(III)-N(His-Im) stretch. The slow form of the enzyme possesses greater intensity in RR peaks near 1620 cm-1 which have been previously attributed by others to partial photoreduction of the enzyme. We have quantitated the amount of laser-induced photoreduction in these RR spectra by comparison with the spectra of mixed-valence derivatives of the enzyme and find that these 1620-cm-1 features are unreliable indicators of photoreduction. The spectra of the fast- and slow-reacting species in H2O and D2O have been compared. The fast-reacting form exhibits a 4-cm-1 shift, from 223 to 219 cm-1, upon transferring to D2O in a peak which we assign as the cytochrome a3 Fe(III)-N(His-Im) stretch. There is a parallel shift in the feature at 1651 cm-1 due to the C = O stretch of the formyl group of cytochrome a. These deuterium shifts are not observed in the slow form.(ABSTRACT TRUNCATED AT 250 WORDS)