Active site of bovine adrenocortical cytochrome P-450(11) beta studied by resonance Raman and electron paramagnetic resonance spectroscopies: distinction from cytochrome P-450scc

Cytochrome P-45011 beta was purified as the 11-deoxycorticosterone-bound form from bovine adrenocortical mitochondria and its active site was investigated by resonance Raman and EPR spectroscopies. Resonance Raman spectra of the purified sample revealed that the heme iron adopts the pure pentacoordinated ferric high-spin state on the basis of the nu 10 (1629cm-1) and nu 3 (1490 cm-1) mode frequencies, which are higher than those of the hexacoordinated ferric high-spin cytochrome P-450scc-substrate complexes. In the ferrous-CO state, a Fe2(+)-CO stretching mode was identified at 481.5 cm-1 on the basis of an isotopic substitution technique; this frequency is very close to that of cytochrome P-450scc in the cholesterol-complexed state (483 cm-1). The EPR spectra of the purified sample at 4.2 K showed ferric high-spin signals (at g = 7.98, 3.65, and 1.71) that were clearly distinct from the cytochrome P-450scc ferric high-spin signals (g = 8.06, 3.55, and 1.68) and confirmed previous assignments of ferric high-spin signals in adrenocortical mitochondria. The EPR spectra of the nitric oxide (NO) complex of ferrous cytochrome P-45011 beta showed EPR signals with rhombic symmetry (gx = 2.068, gz = 2.001, and gy = 1.961) very similar to those of the ferrous cytochrome P-450scc-NO complex in the presence of 22(S)-hydroxycholesterol and 20(R),22-(R)-dihydroxycholesterol at 77 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Electron paramagnetic resonance of the excited triplet state of metal-free and metal-substituted cytochrome c.

The photoactivated metastable triplate states of the porphyrin (free-base, i.e., metal-free) zinc and tin derivatives of horse cytochrome c were investigated using electron paramagnetic resonance. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range 3.8-150 K are discussed in terms of porphyrin-protein interactions. The zero-field splitting parameters D for the free-base, Zn and Sn derivatives are 465 x 10(-4), 342 x 10(-4) and 353 x 10(-4) cm-1, respectively, and are temperature invariant over the temperature ranges studied. AN E value at 4 K of 73 x 10(-4) cm-1 was obtained for Zn cytochrome c, larger than any previously found for Zn porphyrins derivatives of hemeproteins, showing that the heme site of cytochrome c imposes an asymmetric field. Though the E value for Zn cytochrome c is large, the geometry of the site appears quite constrained, as indicated by a spectral line shape showing a single species. Intersystem crossing occurred predominantly to the T2 > zero-field spin sublevel. EPR line shape changes with respect to temperature of Zn cyt c are interpreted in terms of vibronic coupling, and a maximum Jahn-Teller crystal-field splitting of approximately 180 cm-1 is obtained. Sn cytochrome c in comparison with the Zn protein exhibits a photoactivated triplet line shape that is less well resolved in the X-Y region. The magnitude of E value is approximately 60 x 10(-4) cm-1 at 4 K; its value rapidly tends toward zero with increasing temperature, from which a value for the Jahn-Teller crystal-field splitting of > or = 40 cm-1 is estimated. In contrast to those for the metal cytochromes, the magnitude of E value for the free-base derivative was essentially zero at all temperatures studied. This finding is discussed as a consequence of an excited-state tautomerization process that occurs even at 4 K.     

Evidence for structural heterogeneities and a study of exchange coupling. M?ssbauer studies of cytochrome c1aa3 from Thermus thermophilus

We have studied samples of oxidized (as isolated) cytochrome c1aa3 from Thermus thermophilus in the pH range 5.7 to 9.3 with M?ssbauer spectroscopy. In this pH range, the spectra of cytochromes c1 and a are independent of pH, whereas the spectra of cytochrome a3 are not. Most importantly, spectra taken in applied fields up to 6.0 T reveal the presence of multiple ferric forms of cytochrome a3. At any given pH value, at least two high-spin ferric cytochrome a3 species can be distinguished; in addition, most samples contain a low-spin ferric cytochrome a3 species (less than 20% of cytochrome a3). The M?ssbauer spectra show clearly that all forms of cytochrome a3 are spin coupled (to CuB). We have analyzed the high field (H greater than or equal to 1.5 tesla) spectra of a sample at pH 6.5 in the framework of a model that considers isotropic exchange-coupling, JS1.S2, between a high-spin ferric (S1 = 5/2) cytochrome a3 and cupric CuB (S2 = 1/2). In strong applied fields, the spectra can be fitted with any value for J greater than or equal to 0.5 cm-1. In the strong coupling case (J/D1 approximately greater than 3), a zero field splitting parameter D1 approximately 2.5 cm-1 is required for cytochrome a3; this value is distinctly smaller than those observed for high-spin ferric hemes (4-20 cm-1). A model assuming weak coupling magnitude of J approximately 1 cm-1, yields D1 approximately 8 cm-1 and a parameter set for cytochrome a3 quite similar to that reported for metmyoglobin. A J-value of approximately 1 cm-1 does not demand the presence of a ligand bridging between cytochrome a3 and CuB.     

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 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).     

Resonance raman spectroscopy of an ultraviolet-sensitive insect rhodopsin

We present the first visual pigment resonance Raman spectra from the UV-sensitive eyes of an insect, Ascalaphus macaronius (owlfly). This pigment contains 11-cis-retinal as the chromophore. Raman data have been obtained for the acid metarhodopsin at 10 degrees C in both H2O and D2O. The C = N stretching mode at 1660 cm-1 in H2O shifts to 1631 cm-1 upon deuteriation of the sample, clearly showing a protonated Schiff base linkage between the chromophore and the protein. The structure-sensitive fingerprint region shows similarities to the all-trans-protonated Schiff base of model retinal chromophores, as well as to the octopus acid metarhodopsin and bovine metarhodopsin I. Although spectra measured at -100 degrees C with 406.7-nm excitation, to enhance scattering from rhodopsin (lambda max 345 nm), contain a significant contribution from a small amount of contaminants [cytochrome(s) and/or accessory pigment] in the sample, the C = N stretch at 1664 cm-1 suggests a protonated Schiff base linkage between the chromophore and the protein in rhodopsin as well. For comparison, this mode also appears at approximately 1660 cm-1 in both the vertebrate (bovine) and the invertebrate (octopus) rhodopsins. These data are particularly interesting since the absorption maximum of 345 nm for rhodopsin might be expected to originate from an unprotonated Schiff base linkage. That the Schiff base linkage in the owlfly rhodopsin, like in bovine and in octopus, is protonated suggests that a charged chromophore is essential to visual transduction.     

An EPR study of the lineshape of copper in cytochrome c oxidase.

The EPR spectrum of copper in cytochrome c oxidase (EC 1.9.3.1) has been studied between 5 and 220 degreesK, and the spectral parameters have been determined for both forms of EPR-detectable copper by computer simulation methods. Numerical methods have been developed to separate the spectra of intrinsic copper and inactive copper. Evidence is presented to show that inactive copper is probably formed by denaturation. The EPR parameters for intrinsic copper were determined as gx = 1.99, gy = 2.03, gz = 2.185, / Ax(Cu) / = 0.0020 cm-1, / Ay(Cu) / = 0.0025 cm-1, / Az(Cu) / = 0.0030 cm-1. The principal values of the g tensor and the small value of /Az(Cu) / are interpreted in terms of mixing of 3d, 4s, and 4p metal orbitals. A flattened-tetrahedral stereochemistry about Cu2+ with an additional rhombic distrotion is in best agreement with all of the data. The peak-to-peak linewidth is found to be orientation dependent, and is described by a tensor with principal values deltaHx = 45G, deltaHy = 65 G, deltaHz = 85 G. A weak dipolar interaction with a low-spin ferric species stereochemistry for the copper ion is consistent with the electron transport function of the enzyme. Broad EPR signals with a very short spin-lattice relaxation time has been observed near g = 14 and g = 3 at 5 degrees K in oxidized cytochrome oxidase but not in the reduced or denatured enzyme. The possibility that these are due to the "EPR-undetectable" iron and copper is raised.     

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.     

Multiple frequency EPR studies on three forms of oxidized cytochrome c oxidase

Bovine heart mitochondrial cytochrome c oxidase (cytochrome aa3) (EC 1.9.3.1) has been demonstrated to occur in several forms when the redox centers in the protein are thought to be fully oxidized. We report here the results of extensive EPR studies at 3, 8.9, 9.2, 9.4, 15 and 34 GHz on the resting state, the alternative resting state (with g = 12 at 9 GHz) and pulsed state (with g = 5 signal at 9 GHz). Theoretical consideration is given to all binary spin-coupling possibilities under the constraint that the iron atoms are either ferric or ferrous and the copper atoms are either cupric or cuprous. We conclude that the g = 12 signal can arise from any spin system with S greater than 1 and D = 0.15 cm-1. The g = 5 signals originate from an excited, integer-spin system with D = 0.035 cm-1, which is approximately 7 cm-1 above the ground state (not observed in EPR). It is pointed out that in interpretations of data and elaboration of suitable models in this field, the implications of spin-coupling should be considered in a comprehensive and not in a selective way. At 3 GHz, EPR spectra of CuA in the resting, pulsed and anaerobically oxidized states show that this center is identical in its EPR for all three states.     

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.     

Effects of cholesterol and adrenodoxin binding on the heme moiety of cytochrome P-450scc: a resonance Raman study

The effects of cholesterol and adrenodoxin binding on resonance Raman spectra of cytochrome P-450scc in both oxidized and CO-reduced states were examined. Upon cholesterol binding, oxidized cytochrome P-450scc showed a significant shift of spin equilibrium from low-spin to high-spin state. Addition of adrenodoxin caused a complete conversion of cholesterol-bound oxidized cytochrome P-450scc to a pure high-spin state that was considered to be in the hexacoordinated state judged by the v10 mode at 1620 cm-1 and v3 mode around 1485 cm-1. Cholesterol in substrate binding site may oppose a linear and perpendicular binding of carbon monoxide to the reduced heme iron, leading to the distorted Fe-C-O linkage. This is based on the following observations: (1) an increase of the Fe-CO stretching frequency to 483 from 477 cm-1 upon addition of cholesterol; (2) an enhanced photodissociability of bound carbon monoxide of CO complex of cytochrome P-450scc in the presence of cholesterol. As another aspect of the effect of cholesterol on the CO complex form of cytochrome P-450scc, the enhanced stability of the native form ("P-450" form) was observed. There was no additional effect of reduced adrenodoxin on the Raman spectra of the CO-reduced form of cytochrome P-450scc.     

Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureofaciens

Pseudomonas aureofaciens truncates the respiratory reduction of nitrate (denitrification) at the level of N2O. The nitrite reductase from this organism was purified to apparent electrophoretic homogeneity and found to be a blue copper protein. The enzyme contained 2 atoms of copper/85 kDa, both detectable by electron paramagnetic resonance (EPR) spectroscopy. The protein was dimeric, with subunits of identical size (40 +/- 3 kDa). Its pI was 6.05. The EPR spectrum showed an axial signal g at 2.21(8) and g at 2.04(5). The magnitude of the hyperfine splitting (A parallel = 6.36 mT) indicated the presence of type 1 copper only. The electronic spectrum had maxima at 280 nm, 474 nm and 595 nm (epsilon = 7.0 mM-1 cm-1), and a broad shoulder around 780 nm. A copper protein of low molecular mass (15 kDa), with properties similar to azurin, was also isolated from P. aureofaciens. The electronic spectrum of this protein showed a maximum at 624 nm in the visible range (epsilon = 2.5 mM-1 cm-1) and pronounced structures in the ultraviolet region. The EPR parameters were g parallel = 2.26(6) and g perpendicular = 2.05(6), with A parallel = 5.8 mT. The reduced azurin transferred electrons efficiently to nitrite reductase; the product of nitrite reduction was nitric oxide. The specific nitrite-reducing activity with ascorbate-reduced phenazine methosulfate as electron donor was 1 mumol substrate min-1 mg protein-1. The reaction product again was nitric oxide. Nitrous oxide was the reaction product from hydroxylamine and nitrite and from dithionite-reduced methyl viologen and nitrite. No 'oxidase' activity could be demonstrated for the enzyme. Our data disprove the presumed exclusiveness of cytochrome cd1 as nitrite reductase within the genus Pseudomonas.     

Cytochrome c2 and reaction center of Rhodospeudomonas spheroides Ga. membranes. Extinction coefficients, content, half-reduction potentials, kinetics and electric field alterations.

The reduced minus oxidized extinction coefficients (delta epsilon-red-ox) of reaction center P605 when in the chromatophore is about 20% smaller than in the detergent-isolated state. Presumably the coupling of the reaction center protein to the antenna bacteriochlorophylls and carotenoids causes this hypochromism. The chromatophore values for P605 are 19.5 mM- minus 1 times cm- minus 1 with the spectrophotometer on single beam mode at 605 nm, and 29.8 mM- minus 1 times cm- minus 1 on dual wavelength mode set at 605--540 nm. Cytochrome c2, which is not affected by detergent, has a delta epsilon-red-ox value at 550--540 nm of 19.0 mM- minus 1 times cm- minus 1.2. The total bacteriochlorophyll to reaction center bacteriochlorophyll protein (P) ratio is about 100: 1. The cytochrome c2: reaction center protein ratio approaches 2. In current French press chromatophore preparations, about 70% of the reaction centers are each associated on a rapid kinetic basis with two cytochrome c2 molecules (intact P-c2 units). The remaining reaction center proteins are not associated with cytochrome c2 on a kinetically viable bais and may be the result of damage incurred during mechanical rupture of the cells. 3...     

Characterization by FTIR spectroscopy of the photoreduction of the primary quinone acceptor QA in photosystem II.

Molecular changes associated with the photoreduction of the primary quinone acceptor Qa of photosystem II have been characterized by Fourier transform infrared spectroscopy. This reaction was light-induced at room temperature on photosystem II membranes in the presence of hydroxylamine and diuron. A positive signal at 1478 cm-1 is assigned to the C---O stretching mode of the semiquinone anion, and can be correlated to the negative C=O mode(s) of the neutral QA at 1645 cm-1 and/or 16 cm-1. Analogies with bacterial reaction center are found in the amide I absorption range at 1672 cm-1, 1653 cm-1 and 1630 cm-1. The stabilization of QA- does not result from a large protein conformation change, but involves perturbations of several amino acid vibrations. At 1658 cm-1, a negative feature sensitive to 1H-2H exchange is tentatively assigned to a NH2 histidine mode, while tryptophan D2252 could contribute to the signal at 1560/1550 cm-1.     

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)     

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.     

Dynamic interactions of CO with a3Fe and CuB in cytochrome c oxidase in beef heart mitochondria studied by Fourier transform infrared spectroscopy at low temperatures

Carbon monoxide bound to cytochrome c oxidase has been observed by Fourier transform infrared spectroscopy between 10 K and 280 K in the dark and during and after continuous photolysis. CO bound to a3Fe absorbs near 1963 cm-1, with minor bands at lower frequencies. Photolysis at low temperatures transfers CO to CuB, with the major component near 2062 cm-1 and a minor one near 2043 cm-1. Vibrational absorptions are assigned by comparison with heme and copper carbonyls, by frequency dependence of all bands on the isotopic mass of CO, and by similar behavior of major and minor components with photolysis and relaxation kinetics as a function of temperature. Reformation of a3FeCO after photolysis is an apparent first order process below 210 K with a distribution of rate constants. The kinetics are well described by a power law. Arrhenius behavior is followed between 140 K and 180 K to yield a peak activation enthalpy of 40.3 kJ/mol and a distribution in g(H) = 2.56 kJ/mol (full width at half-maximum). The major component of a3FeCO shows a very narrow CO absorption band (full width at half-maximum = 2.4 cm-1), while that of CuBCO shows a broader CO absorption (full width at half-maximum = 6 cm-1). These data indicate that in the reduced carbon monoxide complex, a3FeCO is in highly ordered nonpolar surroundings sufficiently separated from CuB that it is not perturbed by motion of the latter, while CuBCO is in less ordered, more flexible surroundings.     

Bacteriorhodopsin's M412 intermediate contains a 13-cis, 14-s-trans, 15-anti-retinal Schiff base chromophore

The structure of the retinal chromophore about the C = N and C14-C15 bonds in bacteriorhodopsin's M412 intermediate has been determined by analyzing resonance Raman spectra of 2H and 13C isotopic derivatives. Normal mode calculations on 13-cis-retinal Schiff bases demonstrate that the C15-D rock and N-CLys stretch are strongly coupled for C = N-syn chromophores and weakly coupled for C = N-anti chromophores. When the Schiff base geometry is anti, the C15-D rock appears as a localized resonance Raman active mode at approximately 980 cm-1, which is moderately sensitive to 13C substitution at positions 14 and 15 (approximately 7 cm-1) and insensitive to 13C substitution at the epsilon position of lysine. When the Schiff base geometry is syn, in-phase and out-of-phase combinations of the C15-D rock and N-CLys stretch are predicted at approximately 1060 and approximately 910 cm-1, respectively. The in-phase mode is more sensitive to 13C substitution at positions 14 and 15 (approximately 15 cm-1) and at the epsilon position of lysine (approximately 4 cm-1). Calculations and comparison with experimental data on dark-adapted bacteriorhodopsin indicate that the in-phase mode at approximately 1060 cm-1 carries the majority of the resonance Raman intensity. M412 exhibits a C15-D rock at 968 cm-1 that shifts 8 cm-1 when 13C is added at positions 14 and 15 and is insensitive to 13C substitution at the epsilon-position of lysine. This demonstrates that M412 contains a C = N-anti Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS)