Infrared spectra of carbon monoxide complexes of indoleamine 2,3-dioxygenase and L-tryptophan 2,3-dioxygenases. Effects of substrates on the CO-stretching frequencies

Carbonmonoxy indoleamine 2,3-dioxygenase from rabbit small intestine exhibited two CO stretch bands at 1953 and 1933 cm-1 with half-band widths (delta v 1/2) of both approximately 15 cm-1. Upon addition of an excess amount of L-tryptophan, the substrate, the spectrum changed into that with an intense single band at 1902 cm-1 with the delta v 1/2 of 15 cm-1. Carbonmonoxy L-tryptophan 2,3-dioxygenase of Pseudomonas acidovorans in the absence of L-tryptophan showed a fused CO stretch band which consists of two components at 1965 and 1958 cm-1 (delta v 1/2 for the fused band; 25 cm-1), which was converted into a sharp single band at 1968 cm-1 (delta v 1/2; 10 cm-1) upon addition of excess L-tryptophan. On the other hand, CO complex of rat liver L-tryptophan 2,3-dioxygenase in the absence of L-tryptophan gave a spectrum with a poorly defined peak around 1961 cm-1. By the addition of L-tryptophan, the spectrum changed into that with two distinct bands at 1972 and 1920 cm-1 (delta v 1/2; 6 and 13 cm-1, respectively). These spectra were insensitive to pH in a range where the enzymes were not denatured (neutral to near pH 9). The infrared spectra of the carbonmonoxy enzymes were also affected by the addition of certain effectors such as skatole and alpha-methyl-DL-tryptophan, which facilitate the binding of L-tryptophan to the catalytic site of intestinal and Pseudomonas enzymes, respectively. However, the changes were of different types from those by the saturating amount of L-tryptophan. Possible mechanisms for these phenomena are discussed in relation to the structure of the heme-CO complex in these heme-containing dioxygenases.     

Evidence for a band III analogue in the near-infrared absorption spectra of cytochrome c oxidase.

Ground state near-infrared absorption spectra of fully reduced unliganded and fully reduced CO (a2+ CuA+ a3(2+)-CO CuB+) cytochrome c oxidase were investigated. Flash-photolysis time-resolved absorption difference spectra of the mixed-valence (a3+ CuA2+ a3(2+)-CO CuB+) and the fully reduced CO complexes were also studied. A band near 785 nm (epsilon approximately 50 M-1cm-1) was observed in the fully reduced unliganded enzyme and the CO photoproducts. The time-resolved 785 nm band disappeared on the same timescale (t1/2 approximately 7 ms) as CO recombined with cytochrome a3(2+). This band, which is attributed to the unliganded five coordinate ferrous cytochrome a3(2+), has some characteristics of band III in deoxy-hemoglobin and deoxy-myoglobin. A second band was observed at approximately 710 nm (epsilon approximately 80 M-1cm-1) in the fully reduced unliganded and the fully reduced CO complexes. This band, which we assign to the low spin ferrous cytochrome a, appears to be affected by the ligation state at the cytochrome a3(2+) site.     

Unusually strong H-bonding to the heme ligand and fast geminate recombination dynamics of the carbon monoxide complex of Bacillus subtilis truncated hemoglobin

The active site of the oxygen-avid truncated hemoglobin from Bacillus subtilis has been characterized by infrared absorption and resonance Raman spectroscopies, and the dynamics of CO rebinding after photolysis has been investigated by picosecond transient absorption spectroscopy. Resonance Raman experiments on the CO bound adduct revealed the presence of two Fe-CO stretching bands at 545 and 520 cm-1, respectively. Accordingly, two C-O stretching bands at 1924 and 1888 cm-1 were observed in infrared absorption and resonance Raman measurements. The very low C-O stretching frequency at 1888 cm-1 (corresponding to the extremely high RR stretching frequency at 545 cm-1) indicates unusually strong hydrogen bonding between CO and distal residues. On the basis of a comparison with other truncated hemoglobin it is envisaged that the two CO conformers are determined by specific interactions with the TrpG8 and TyrB10 residues. Mutation of TrpG8 to Leu deeply alters the hydrogen-bonding network giving rise mainly to a CO conformer characterized by a Fe-CO stretching band at 489 cm-1 and a CO stretching band at 1958 cm-1. Picosecond laser photolysis experiments carried out on the CO bound adduct revealed dynamical processes that take place within a few nanoseconds after photolysis. Picosecond dynamics is largely dominated by CO geminate rebinding and is consistent with strong H-bonding contributions of TyrB10 and TrpG8 to ligand stabilization.     

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.     

On the structures of flavoprotein D-amino acid oxidase purple intermediates. A resonance Raman study

Resonance Raman (RR) spectra were obtained in H2O or D2O solution for the purple intermediates of D-amino acid oxidase (DAO) with isotopically labeled substrates, i.e., [1-13C]-, [2-13C]-, [3-13C]-, [15N]-, and [3,3,3-D3]alanine; [carboxyl-13C]- and [15N]proline. RR spectra were also measured for the intermediates of DAO reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]FAD in D2O. The isotopic shift of the 1692 cm-1 band upon [15N]- or [2-13C]-substitution of alanine indicates that the band is due to the C = N stretching mode of an imino acid derived from D-alanine, i.e., alpha-iminopropionate. The 1658 cm-1 band with D-proline was also assigned to the C = N stretching mode of an imino acid derived from D-proline, i.e., delta 1-pyrrolidine-2-carboxylate, since the band shifts to 1633 cm-1 upon [15N]-substitution and its stretching frequency is generally found in this frequency region. Since the band shifts to low frequency in D2O, the imino acid should have a protonated imino group such as the C = N+1H form. The intense band at 1363 cm-1 with D-alanine was assigned to a mixing of the CO2- symmetric stretching and CH3 symmetric deformation modes in alpha-iminopropionate, based on the isotope effects. The 1359 cm-1 band with D-proline has probably contributions of CO2- symmetric stretching and CH2 wagging, considering the isotope effects with [carboxyl-13C]proline. The 1359 cm-1 band with D-proline was split into 1371 cm-1 and 1334 cm-1 bands in D2O. As this splitting of the 1359 cm-1 band with D-proline in D2O can not be interpreted only by the replacement of the C = N+1-H proton by deuterium, the carboxylate of the imino acid probably interacts with the enzyme through some proton(s) exchangeable by deuterium(s) in D2O. The bands around 1605 cm-1 which shift upon [4a-13C]- and [4,10a-13C2]-labeling of FAD are derived from a fully reduced flavin, because the isotopic shifts of the band are very different from those of the bands of oxidized or semiquinoid flavin observed near 1605 cm-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

A resonance Raman study on a reaction intermediate of Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating).

Resonance Raman (RR) spectra of purple intermediates of L-phenylalanine oxidase (PAO) with non-labeled and isotopically labeled phenylalanines as substrates, i.e., [1-13C], [2-13C], [ring-U-13C6], and [15N]phenylalanines, were measured with excitation at 632.8 nm within the broad absorption band around 540 nm. The spectra obtained resemble those of purple intermediates of D-amino acid oxidase (DAO). The isotope effects on the 1,665 cm-1 band with [15N] or [2-13C]phenylalanine indicate that the band is due to the C = N stretching mode of an imino acid derived from phenylalanine, i.e., alpha-imino-beta-phenylpropionate. The intense band at 1,389 cm-1 is contributed to by the CO2- symmetric stretching and C-CO2- stretching modes of alpha-imino-beta-phenylpropionate. The 1,602 cm-1 band, which does not shift upon isotopic substitution of phenylalanine, corresponds to the 1,605 cm-1 band of DAO purple intermediates and was assigned to a vibrational mode associated with the C(10a) = C(4a) - C(4) = O moiety of reduced flavin. These results confirm that PAO purple intermediates consist of the reduced enzyme and an imino acid derived from a substrate, and suggest that the plane defined by C(10a) = C(4a) - C(4) = O of reduced flavin and the plane containing H2+N = C - CO2- of an imino acid are arranged in close contact to each other, generating a charge-transfer interaction.     

Infrared evidence of cyanide binding to iron and copper sites in bovine heart cytochrome c oxidase. Implications regarding oxygen reduction

Cyanide binding to bovine heart cytochrome c oxidase at five redox levels has been investigated by use of infrared and visible-Soret spectra. A C-N stretch band permits identification of the metal ion to which the CN- is bound and the oxidation state of the metal. Non-intrinsic Cu, if present, is detected as a cyanide complex. Bands can be assigned to Cu+CN at 2093 cm-1, Cu2+CN at 2151 or 2165 cm-1, Fe3+CN at 2131 cm-1, and Fe2+CN at 2058 cm-1. Fe2+CN is found only when the enzyme is fully reduced whereas the reduced Cu+CN occurs in 2-, 3-, and 4-electron reduced species. A band for Fe3+CN is not found for the complex of fully oxidized enzyme but is for all partially reduced species. Cu2+CN occurs in both fully oxidized and 1-electron-reduced oxidase. CO displaces the CN- at Fe2+ to give a C-O band at 1963.5 cm-1 but does not displace the CN- at Cu+. Another metal site, noted by a band at 2042 cm-1, is accessible only in fully reduced enzyme and may represent Zn2+ or another Cu+. Binding of either CN- or CO may induce electron redistribution among metal centers. The extraordinary narrowness of ligand infrared bands indicates very little mobility of the components that line the O2 reduction site, a property of potential advantage for enzyme catalysis. The infrared evidence that CN- can bind to both Fe and Cu supports the possibility of an O2 reduction mechanism in which an intermediate with a mu-peroxo bridge between Fe and Cu is formed. On the other hand, the apparent independence of Fe and Cu ligand-binding sites makes a heme hydroperoxide (Fe-O-O-H) intermediate an attractive alternative to the formation an Fe-O-O-Cu linkage.     

Effects of crystallization on the heme-carbon monoxide moiety of bovine heart cytochrome c oxidase carbonyl.

Cytochrome c oxidase isolated from bovine heart was crystallized in the fully reduced carbon monoxide (CO)-bound form. To evaluate the structure of the O2 reaction site in crystals and in solution, the bound C-O stretch infrared band in protein crystals was compared with the band for protein solution. In solution, the C-O stretch band could be deconvoluted into two extremely narrow bands, one at 1963.6 cm-1 with delta v1/2 = 3.4 cm-1 of 60% Gaussian/40% Lorentzian character represented 86% of the total band area and the other at 1960.3 cm-1 with delta v1/2 = 3.0 cm-1 of 47% Gaussian/53% Lorentzian character represented 14% of the total band area. The crystals exhibited two deconvoluted C-O infrared bands having very similar band parameters with those in solution. These findings support the presence of two structurally similar conformers in both crystals and solution. Thus crystallization of this enzyme does not affect the structure at the CO-binding site to as great extent as has been noted for myoglobin and hemoglobin carbonyls, indicating that the active (CO- or O2-binding) site of cytochrome c oxidase must be conformationally very stable and highly ordered compared to other hemoproteins such as hemoglobin.     

An FT-IR study of DNA and RNA conformational transitions at low temperatures

Fourier Transform Infrared (FT-IR) spectra of solid samples of DNA and RNA obtained from freeze-drying at solid CO2 and liquid nitrogen temperatures, have been recorded and correlation between the conformational transitions and spectral changes is proposed. It is concluded that an equilibrium exists between A, B and Z conformations at low temperatures for the DNA molecule, which is temperature dependent, whereas the RNA molecule exhibits only the A conformation. The results have been compared with the metal-adducts of DNA and RNA, where one of the conformations is predominant. Marker infrared bands for the B conformer have been found to be the strong band at 825 cm-1 (sugar conformer mode) and a band with medium intensity at 690 cm-1 (guanine breathing mode). The A conformation showed characteristic bands at 810 and 675 cm-1. The B to Z conformational transition was characterized by the strong absorption bands near 820-810 cm-1 and at 665-600 cm-1.     

Reversible acidic-alkaline transition of the carbon monoxide complex of cytochrome c peroxidase

The Soret absorption band of the ferrous carbon monoxide (CO) complex of cytochrome c peroxidase exhibited a blue shift from 423.7 to 420 nm upon an increase in pH from 6.5 to 8.5. The spectral change was reversible with an isosbestic point at 422 nm. The pH dependence of this spectral change gave a sigmoidal curve fitted well to a theoretical curve of a cooperative release of two protons with a pK value of 7.5, indicating the existence of the acidic and alkaline forms of the ferrous CO enzyme. Upon irradiation of light flash (100 J of power and 30-microseconds), the heme-bound CO was readily dissociated in both acidic and alkaline forms with a quantum yield of approximately unity. On the other hand, the rate of recombination of the dissociated CO with the heme iron was significantly different between these two forms; the recombination rate constants were 1.1 X 10(3) and 3.0 X 10(4) M-1 S-1 at 25 degrees C for the acidic and alkaline forms, respectively. At intermediate pH values, kinetics of recombination were biphasic, consisting of the slow and fast processes with the appropriate rate constants mentioned above. When the fraction of the fast process was plotted against pH, the pH profile coincided with the spectrophotometric pH titration curve described above. Thus, it was concluded that the acidic and alkaline forms of the enzyme were responsible for the slow and fast processes, respectively. In infrared spectroscopy, the acidic form showed a narrow CO stretching band at 1922 cm-1 with a half-band width of 12.5 cm-1, while the alkaline form exhibited a broad CO-stretching band at 1948 cm-1 with a half-band width of 33 cm-1. Significance of these results are discussed in relation to the structure of the heme vicinity on the CO complex of cytochrome c peroxidase.     

Resonance Raman evidence that distal histidine protonation removes the steric hindrance to upright binding of carbon monoxide by myoglobin

The resonance Raman band assigned to Fe--CO stretching in the sperm whale myoglobin CO adduct shifts from 507 cm-1 at neutral pH to 488 cm-1 at low pH, in concert with a shift of the C-O stretching infrared band from 1947 to 1967 cm-1 (Fuchsman & Appleby, 1979), while the 575-cm-1 Fe-C-O bending RR band loses intensity. The pKa that characterizes these changes is approximately 4.4. The vibrational frequencies at low pH are well modeled by the protein-free CO, imidazole adduct of protoheme in a nonpolar solvent while those at high pH are modeled by the adduct of a heme with a covalent strap (Yu et al., 1983) which inhibits upright CO binding. It is inferred that the Fe-C-O unit changes from a tilted to an upright geometry when the distal histidine is protonated, because its side chain swings out of the heme pocket due to electrostatic repulsion with a nearby arginine residue. A different protonation step (pKa = 5.7), which has been shown to modulate the CO rebinding kinetics (Doster et al., 1982) as well as the optical spectrum (Fuchsman & Appleby, 1979), is suggested to involve a global structure change associated with protonation of histidine residues distant from the heme.     

Buffer-anion-dependent Ca2+ leaching from horseradish peroxidase at low pH

Despite highly conserved active-site structures, members of the plant peroxidase superfamily exhibit a wide range of pH optima. Horseradish peroxidase isozyme C (HRPC) is an ideal peroxidase to investigate the structural determinants of pH stability and activity in superfamily members. Conflicting reports exist on the low-pH stability of HRPC and consequently the pKa of the catalytic distal histidine, which is neutral in active peroxidases. Towards resolving such discrepancies, acid-induced changes in HRPC from two popular commercial suppliers were systematically analyzed. Specifically, FTIR v(CO) and Soret-CD spectra of HRPC-CO and Soret absorption of ferric HRPC were recorded to probe time-dependent heme-pocket changes at pH 3.0 in phosphate, citrate and formate buffers, while the FTIR amide I' and far-UV CD spectra were examined to probe changes in secondary structure. Both HRPC-CO samples exhibited identical pH 7.0 v(CO) bands at 1934 and 1905 cm-1. In the pH 3.0 spectrum of sample A, the 1934 cm-1 band was dominant while a broad 1969 cm-1 band appeared in sample B. The intensity of this band, which is assigned to solvent-exposed heme, was greater in citrate than phosphate buffer, but in formate the 1934 cm-1 band remained dominant. Other spectral changes mirrored the v(CO) trends. No time- or buffer-anion-dependent conformation changes were detected in 1 mM CaCl2, revealing that buffer-anion-dependent leaching of stabilizing Ca2+ from HRPC occurs at pH 3.0. Since the N-glycans present in HRPC are of the flexible protein-surface-shielding type, the variation in low-pH conformational stability of the HRPC samples could be attributed to heterogeneous glycosylation, which was detected by SDS-PAGE. It is further proposed that glycosylation patterns may affect the low-pH stability of class II and III plant peroxidases.     

Alternative carbon monoxide binding modes for horseradish peroxidase studied by resonance Raman spectroscopy

Resonance Raman (RR) spectroscopy and infrared spectroscopy have been used to characterize the three vibrational modes, CO and FeC stretching and FeCO bending, for carbon monoxide bound to reduced horseradish peroxidase, with the aid of 13CO and C18O isotope shifts. At high pH, one species, I, is observed, with nu FeC = 490 cm-1 and nu CO = 1932 cm-1. The absence of a band attributable to delta FeCO suggests a linear FeCO unit normal to the heme plane. The data were consistent with I having a strongly H-bonded proximal histidine, as shown by a comparison with imidazole and imidazolate adducts of FeIIPPDME(CO) (PPDME = protoporphyrin IX dimethyl ester), with nu FeC = 497 and 492 cm-1 and nu CO = 1960 and 1942 cm-1. At low pH an additional species, II, is observed, with nu FeC = 537 cm-1, nu CO = 1904 cm-1, and delta FeCO = 587 cm-1; it is attributed to FeCO that is H bonded to a protonated distal histidine, the H bond strongly lowering nu CO and raising nu FeC. The appearance of delta FeCO in the RR spectrum suggests that the FeCO unit in II is tilted with respect to the heme plane. At low pH, the population of I and II depends on the CO concentration. I dominates at low CO/protein levels but is replaced by II as the amount of CO is increased. This behavior is suggested to arise from secondary binding of CO, which induces a conformation change involving the distal residues of the heme pocket.     

A resonance Raman study on the structures of complexes of flavoprotein D-amino acid oxidase

Resonance Raman (RR) spectra were obtained for the purple complexes of D-amino acid oxidase (DAO) with D-lysine or N-methylalanine. RR spectra of a complex of oxidized DAO with the oxidation product of D-lysine or D-proline were also measured. The isotope shifts of the observed bands of the purple complex with D-lysine upon 13C- or 15N-substitution of lysine indicate that the ligand is delta 1-piperideine-2-carboxylate. That the band at 1671 cm-1 for the purple intermediate with N-methylalanine shifts to 1666 cm-1 in D2O solution indicates that the imino acid, N-methyl-alpha-iminopropionate, has a protonated imino group. Many bands due to a ligand in the RR spectra of the complex of oxidized DAO with an oxidation product can be observed below 1000 cm-1, but no band for the purple complex is seen in this frequency region. The band associated with the CO2-symmetric stretching mode of the product, such as delta 1-piperideine-2-carboxylate or delta 1-pyrrolidine-2-carboxylate, complexed with the oxidized DAO shifts in D2O solution. This suggests that the product imino acid interacts with the enzyme through some proton(s).     

Infrared spectra of carbon monoxide bound to mitochondria from diverse species and tissues reveal structurally similar cytochrome c oxidase dioxygen reaction sites

Infrared bands for CO bound to mitochondria from bovine and porcine hearts, bovine brain, rat kidney, and blowfly flight muscle and to intact blowfly flight muscle have been measured in the carbon-oxygen stretch region. Each spectrum contains a narrow band near 1963 cm-1 similar to the major band found earlier for the carbonyl cytochrome c oxidase purified from bovine heart. A second band near 1959 cm-1 ascribed to a less stable conformer of the purified oxidase carbonyl is also detected in mitochondria. These spectra support very similar CO (and O2) binding sites among all the oxidases examined whether the enzyme is purified or is still within mitochondria or intact tissue and therefore suggest that the reduced heme A ligand binding site has been highly conserved during evolution.     

Structural heterogeneity of the Fe(2+)-N epsilon (HisF8) bond in various hemoglobin and myoglobin derivatives probed by the Raman-active iron histidine stretching mode.

We have examined the Fe(2+)-N epsilon (HisF8) complex in hemoglobin A (HbA) by measuring the band profile of its Raman-active nu Fe-His stretching mode at pH 6.4, 7.0, and 8.0 using the 441-nm line of a HeCd laser. A line shape analysis revealed that the band can be decomposed into five different sublines at omega 1 = 195 cm-1, omega 2 = 203 cm-1, omega 3 = 212 cm-1, omega 4 = 218 cm-1, and omega 5 = 226 cm-1. To identify these to the contributions from the different subunits we have reanalyzed the nu Fe-His band of the HbA hybrids alpha(Fe)2 beta(Co)2 and alpha(Co)2 beta(Fe)2 reported earlier by Rousseau and Friedman (D. Rousseau and J. M. Friedman. 1988. In Biological Application on Raman Spectroscopy. T. G. Spiro, editor, 133-216). Moreover we have reanalyzed other Raman bands from the literature, namely the nu Fe-His band of the isolated hemoglobin subunits alpha SH- and beta SH-HbA, various hemoglobin mutants (i.e., Hb(TyrC7 alpha-->Phe), Hb(TyrC7 alpha-->His), Hb M-Boston and Hb M-Iwate), N-ethylmaleimide-des(Arg141 alpha) hemoglobin (NES-des(Arg141 alpha)HbA) and photolyzed carbonmonoxide hemoglobin (Hb*CO) measured 25 ps and 10 ns after photolysis. These molecules are known to exist in different quaternary states. All bands can be decomposed into a set of sublines exhibiting frequencies which are nearly identical to those found for deoxyhemoglobin A. Additional sublines were found to contribute to the nu Fe-His band of NES-des(Arg141 alpha) HbA and the Hb*CO species. The peak frequencies of the bands are determined by the most intensive sublines. Moreover we have measured the nu Fe-His band of deoxyHbA at 10 K in an aqueous solution and in a 80% glycerol/water mixture. Its subline composition at this temperature depends on the solvent and parallels that of more R-like hemoglobin derivatives. We have also measured the optical charge transfer band III of deoxyHbA at room temperature and found, that at least three subbands are required to fit its asymmetric band shape. This corroborates the findings on the nu Fe-His band in that it is indicative of a heterogeneity of the Fe(2+)-N epsilon(HisF8) bond. Finally we measured the nu Fe-His band of horse heart deoxyMb at different temperatures and decomposed it into three different sublines. In accordance with what was obtained for HbA their intensities rather than their frequencies are temperature-dependent.(ABSTRACT TRUNCATED AT 400 WORDS)     

The resonance Raman frequencies of the Fe-CO stretching and bending modes in the CO complex of cytochrome P-450cam

Resonance Raman spectra of the ferrous CO complex of cytochrome P-450cam have been observed both in its camphor-bound and free states. Upon excitation at 457.9 nm, near the absorption maximum of the Soret band, the ferrous CO complex of the camphor-bound enzyme showed an anomalously intense Raman line at 481 cm-1 besides the strong Raman lines at 1366 and 674 cm-1 for the porphyrin vibrations. The Raman line at 481 cm-1 (of the 12C16O complex) shifted to 478 cm-1 upon the substitution by 13C16O and to 473 cm-1 by 12C18O without any detectable shift in porphyrin Raman lines. This shows that the line at 481 cm-1 is assignable to Fe-CO stretching vibration. By the excitation at 457.9 nm, a weak Raman line was also observed at 558 cm-1, which was assigned to the Fe-C-O bending vibration, because it was found to shift by -14 cm-1 on 13C16O substitution while only -3 cm-1 on 12C18O substitution. These stretching and bending vibrations of the Fe-CO bond were not detected with the excitation at 413.1 nm, though the porphyrin Raman lines at 1366 and 674 cm-1 were clearly observed. When the substrate, camphor, was removed from the enzyme, the Fe-CO stretching vibration was found to shift to 464 cm-1 from 481 cm-1, while no detectable changes were found in porphyrin Raman lines. This means that the bound substrate interacts predominantly with the Fe-CO portion of the enzyme molecule.     

Time-resolved FT-IR studies on the CO adduct of Paracoccus denitrificans cytochrome c oxidase: comparison of the fully reduced and the mixed valence form.

The rebinding of CO to cytochrome c oxidase from Paracoccus denitrificans in the fully reduced and in the half-reduced (mixed valence) form as a function of temperature was investigated using time-resolved rapid-scan FT-IR spectroscopy in the mid-IR (1200-2100 cm-1). For the fully reduced enzyme, rebinding was complete in approximately 2 s at 268 K and showed a biphasic reaction. At 84 K, nonreversible transfer of CO from heme a3 to CuB was observed. Both photolysis at 84 K and photolysis at 268 K result in FT-IR difference spectra which show similarities in the amide I, amide II, and heme modes. Both processes, however, differ in spectral features characteristic for amino acid side chain modes and may thus be indicative for the motional constraint of CO at low temperature. Rebinding of photodissociated CO for the mixed-valence enzyme at 268 K is also biphasic, but much slower as compared to the fully reduced enzyme. FT-IR difference spectra show band features similar to those for the fully reduced enzyme. Additional strong bands in the amide I and amide II range indicate local conformational changes induced by electron and coupled proton transfer. These signals disappear when the temperature is lowered to 84 K. At 268 K, a difference signal at 1746 cm-1 is observed which is shifted by 6 cm-1 to 1740 cm-1 in 2H2O. The absence of this signal for the mutant Glu 278 Gln allows assignment to the COOH stretching mode of Glu 278, and indicates changes of the conformation, proton position, or protonation of this residue upon electron transfer.     

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.     

Cytochrome c peroxidase mutant active site structures probed by resonance Raman and infrared signatures of the CO adducts

Vibrational frequencies associated with FeC and CO stretching and FeCO bending modes have been determined via resonance Raman (RR) and infrared (IR) spectroscopy for cytochrome c peroxidase (CCP) mutants prepared by site-directed mutagenesis. 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. The data were analyzed with the aid of a recently established correlation between nu FeC and nu CO, which can be used to distinguish between back-bonding and axial ligand donor effects. At high pH all adducts showed essentially the same vibrational pattern (form I') with nu FeC approximately 505 cm-1, nu CO approximately 1948 cm-1, and delta FeCO (weak RR band) approximately 576 cm-1. These frequencies are very similar to those shown by the myoglobin CO adduct and imply a "normal" H-bond of the proximal histidine. At pH 7 (pH 6 for Asn-235 and Leu-48), different forms are seen for different proteins: form I (nu FeC approximately 500 cm-1, nu CO = 1922-1941 cm-1, and delta FeCO approximately 580 cm-1, very weak) in the case of CCP(MI) and Phe-191, as well as bakers' yeast CCP, or form II (nu FeC approximately 530 cm-1, nu CO = 1922-1933 cm-1, and delta FeCO = 585 cm-1, moderately strong) for Asn-235 and Phe-51.(ABSTRACT TRUNCATED AT 250 WORDS)