Does superoxide channel between the copper centers in peptidylglycine monooxygenase? A new mechanism based on carbon monoxide reactivity

Peptidylglycine monooxygenase (PHM) carries out the hydroxylation of the alpha-C atom of glycine-extended propeptides, the first step in the amidation of peptide hormones by the bifunctional enzyme peptidyl-alpha-amidating monooxygenase (PAM). Since PHM is a copper-containing monooxygenase, a study of the interaction between the reduced enzyme and carbon monoxide has been carried out as a probe of the interaction of the Cu(I) sites with O(2). The results show that, in the absence of peptide substrate, reduced PHM binds CO with a stoichiometry of 0.5 CO/Cu(I), indicating that only one of the two copper centers, Cu(B), forms a Cu(I)-carbonyl. FTIR spectroscopy shows a single band in the 2200-1950 cm(-)(1) energy region with nu(CO) = 2093 cm(-)(1) assigned to the intraligand C-O stretch via isotopic labeling with (13)CO. A His242Ala mutant of PHM, which deletes the Cu(B) site by replacing one of its histidine ligands, completely eliminates CO binding. EXAFS spectroscopy is consistent with binding of a single CO ligand with a Cu-C distance of 1.82 +/- 0.03 A. The Cu-S(met) distance increases from 2.23 +/- 0. 02 A in the reduced unliganded enzyme to 2.33 +/- 0.01 A in the carbonylated enzyme, suggesting that the methionine-containing Cu(B) center is the site of CO binding. The binding of the peptide substrate N-Ac-tyr-val-gly perturbs the CO ligand environment, eliciting an IR band at 2062 cm(-)(1) in addition to the 2093 cm(-)(1) band. (13)CO isotopic substitution assigns both frequencies as C-O stretching bands. The CO:Cu binding stoichiometry and peptide/CO FTIR titrations indicate that the 2062 cm(-)(1) band is due to binding of CO at a second site, most likely at the Cu(A) center. This suggests that peptide binding may activate the Cu(A) center toward O(2) binding and reduction to superoxide. As a result of these findings, a new mechanism is proposed involving channeling of superoxide across the 11 A distance between the two copper centers.     

Characterization of a half-apo derivative of peptidylglycine monooxygenase. Insight into the reactivity of each active site copper

A derivative of peptidylglycine monooxygenase which lacks the CuH center has been prepared and characterized. This form of the enzyme is termed the half-apo protein. Copper-to-protein stoichiometric measurements establish that the protein binds only one of the two copper centers (CuM and CuH) found in the native enzyme. Confirmation that the methionine-containing CuM has been retained has been obtained from EXAFS experiments which show that the characteristic signature of the Cu-S(Met) interaction is preserved. The half-apo derivative binds 1 equiv of CO per copper with an IR frequency of 2092 cm(-1), and this monocarbonyl also displays the Cu-S(Met) interaction in its EXAFS spectrum. These results allow unambiguous assignment of the 2092 cm(-1) band as a CuM-CO species. Binding of CO in the presence of peptide substrate was also investigated. In the native enzyme, substrate induced binding of a second CO molecule with an IR frequency of 2062 cm(-1), tentatively assigned to a CO complex of the histidine-containing CuH site. Unexpectedly, this reactivity is also observed in the half-apo derivative, although the intensity distribution of the CO stretches now indicates that the copper has been partially transferred to a second site, believed to be CuH. The implications of this observation are discussed in terms of a possible additional peptide binding site close to the CuH center.     

The catalytic copper of peptidylglycine alpha-hydroxylating monooxygenase also plays a critical structural role

Many bioactive peptides require amidation of their carboxy terminus to exhibit full biological activity. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first of the two steps of this reaction, is composed of two domains, each of which binds one copper atom (CuH and CuM). The CuM site includes Met(314) and two His residues as ligands. Mutation of Met(314) to Ile inactivates PHM, but has only a minimal effect on the EXAFS spectrum of the oxidized enzyme, implying that it contributes only marginally to stabilization of the CuM site. To characterize the role of Met(314) as a CuM ligand, we determined the structure of the Met(314)Ile-PHM mutant. Since the mutant protein failed to crystallize in the conditions of the original wild-type protein, this structure determination required finding a new crystal form. The Met(314)Ile-PHM mutant structure confirms that the mutation does not abolish CuM binding to the enzyme, but causes other structural perturbations that affect the overall stability of the enzyme and the integrity of the CuH site. To eliminate possible effects of crystal contacts, we redetermined the structure of wt-PHM in the Met(314)Ile-PHM crystal form and showed that it does not differ from the structure of wild-type (wt)-PHM in the original crystals. Met(314)Ile-PHM was also shown to be less stable than wt-PHM by differential scanning calorimetry. Both structural and calorimetric studies point to a structural role for the CuM site, in addition to its established catalytic role.     

Characterization of a carbon monoxide complex of reduced dopamine beta-hydroxylase. Evidence for inequivalence of the Cu(I) centers

Ascorbate-reduced dopamine beta-hydroxylase (DBH) is inhibited by CO in a competitive manner with respect to molecular O2. Measurement of the stoichiometry of CO binding indicates 0.50 CO bound per Cu(I), which provides the first evidence that the Cu(I) centers in the reduced enzyme are structurally inequivalent. FTIR spectroscopy has been used to detect an infrared absorption band characteristic of coordinated CO, with v(CO) = 2089 cm-1. Comparison of this frequency with those of other Cu(I)-carbonyls in both inorganic and protein systems suggests a coordination site with fewer or less basic ligands than the 3-histidine site of carbon-monoxy hemocyanin.     

The catalytic role of the copper ligand H172 of peptidylglycine alpha-hydroxylating monooxygenase (PHM): a spectroscopic study of the H172A mutant

The spectroscopic characterization of the H172A mutant of peptidylglycine alpha-hydroxylating monooxygenase (PHM) was undertaken to determine the importance of this Cu(H) ligand in the catalytic mechanism of PHM. Mutation of this histidine reduced the activity of the enzyme over 300-fold with little effect on the structure of the oxidized form. However, the reduced enzyme showed a decrease in the average Cu-N(His) distances from 1.96 A in wild-type PHM to 1.89 A in H172A associated with a change in the structure of Cu(H) from distorted T-shaped planar in the wild type to 2-coordinate in the mutant. Binding of CO was retained at the Cu(M) site (similar to wild type), and peptide substrate binding continued to activate a second site for CO binding. Confirmation of this substrate-induced CO binding site at Cu(H) was obtained through the observation that loss of the H172 Cu(H) ligand caused a 3 cm(-)(1) blue shift in the nu(CO) for this copper carbonyl. Possible mechanistic roles for the H172 ligand are discussed.     

Structural changes that occur upon photolysis of the Fe(II)(a3)-CO complex in the cytochrome ba(3)-oxidase of Thermus thermophilus: a combined X-ray crystallographic and infrared spectral study demonstrates CO binding to Cu(B)

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a(3) moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba(3)-oxidase from Thermus thermophilus, determined at ~2.8-3.2? resolution, reveal a Fe-C distance of ~2.0?, a Cu-O distance of 2.4? and a Fe-C-O angle of ~126°. Upon photodissociation at 100K, X-ray structures indicate loss of Fe(a3)-CO and appearance of Cu(B)-CO having a Cu-C distance of ~1.9? and an O-Fe distance of ~2.3?. Absolute FTIR spectra recorded from single crystals of reduced ba(3)-CO that had not been exposed to X-ray radiation, showed several peaks around 1975cm(-1); after photolysis at 100K, the absolute FTIR spectra also showed a significant peak at 2050cm(-1). Analysis of the 'light' minus 'dark' difference spectra showed four very sharp CO stretching bands at 1970cm(-1), 1977cm(-1), 1981cm(-1), and 1985cm(-1), previously assigned to the Fe(a3)-CO complex, and a significantly broader CO stretching band centered at ~2050cm(-1), previously assigned to the CO stretching frequency of Cu(B) bound CO. As expected for light propagating along the tetragonal axis of the P4(3)2(1)2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba(3) crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO(2) at 2337cm(-1) and one from traces of CO at 2133cm(-1); while bands associated with CO bound to either Fe(a3) or to Cu(B) in "light" minus "dark" FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2? and FTIR spectra support the long-held position that photolysis of Fe(a3)-CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu(B) atom; Fe(a3) and Cu(B) ligation, at the resolutions reported here, are otherwise unaltered.     

Supplying copper to the cuproenzyme peptidylglycine alpha-amidating monooxygenase

We explored the role of known copper transporters and chaperones in delivering copper to peptidylglycine-alpha-hydroxylating monooxygenase (PHM), a copper-dependent enzyme that functions in the secretory pathway lumen. We examined the roles of yeast Ccc2, a P-type ATPase related to human ATP7A (Menkes disease protein) and ATP7B (Wilson disease protein), as well as yeast Atx1, a cytosolic copper chaperone. We expressed soluble PHMcc (catalytic core) in yeast using the yeast pre-pro-alpha-mating factor leader region to target the enzyme to the secretory pathway. Although the yeast genome encodes no PHM-like enzyme, PHMcc expressed in yeast is at least as active as PHMcc produced by mammalian cells. PHMcc partially co-migrated with a Golgi marker during subcellular fractionation and partially co-localized with Ccc2 based on immunofluorescence. To determine whether production of active PHM was dependent on copper trafficking pathways involving the CCC2 or ATX1 genes, we expressed PHMcc in wild-type, ccc2, and atx1 mutant yeast. Although ccc2 and atx1 mutant yeast produce normal levels of PHMcc protein, it lacks catalytic activity. Addition of exogenous copper yields fully active PHMcc. Similarly, production of active PHM in mouse fibroblasts is impaired in the presence of a mutant ATP7A gene. Although delivery of copper to lumenal cuproproteins like PAM involves ATP7A, lumenal chaperones may not be required.     

Spectroscopic characterization of carbon monoxide complexes generated for copper/topa quinone-containing amine oxidases

Carbon monoxide complexes have been generated for copper/topa quinone (TPQ)-containing amine oxidases from Arthrobactor globiformis (AGAO) and Aspergillus niger (AO-I) and characterized by various spectroscopic measurements. Addition of CO to AGAO anaerobically reduced with its substrate 2-phenylethylamine led to a slight increase of absorption bands at 440 and 470 nm derived from the semiquinone form (TPQ(sq)) of the TPQ cofactor, concomitantly giving rise to new CO-related absorption bands at 334 and 434 nm. The intensity of the TPQ(sq) radical EPR signal at g = 2.004 also increased in the presence of CO, while its hyperfine coupling structure was affected insignificantly. FT-IR measurements revealed C-O stretching bands (nu(CO)) at 2063 and 2079 cm(-1) for the CO complex of the substrate-reduced AGAO (at 2085 cm(-1) for AO-I), which shifted nearly 100 cm(-1) to lower frequencies upon using (13)C(18)O. Collectively, these results suggest that CO is bound to the Cu(I) ion in the Cu(I)/TPQ(sq) species formed in the reductive half-reaction of amine oxidation, thereby shifting the Cu(II)/aminoresorcinol right arrow over left arrow Cu(I)/semiquinone equilibrium toward the latter. When AGAO was reduced with dithionite, an intermediary form of the enzyme with Cu(II) reduced to Cu(I) but TPQ still in the oxidized state (TPQ(ox)) was produced. Dithionite reduction of AGAO in the presence of CO resulted in the immediate formation of FT-IR bands at 2064 and 2083 cm(-1), which were assigned to the nu(CO) bands of the CO bound to the TPQ(ox) enzyme. The intense 2083 cm(-1) band was then displaced by a new band at 2077 cm(-1), corresponding to the formation of the fully reduced topa. Significant variation of these nu(CO) frequencies indicates that vibrational properties of CO bound to copper amine oxidases are sensitively influenced by the coordination structure of the Cu(I) ion, which may be modulated by the chemical and redox states of the TPQ cofactor.     

Infrared studies of carbon monoxide binding to carbon monoxide dehydrogenase/acetyl-CoA synthase from Moorella thermoacetica

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a bifunctional enzyme that catalyzes the reversible reduction of carbon dioxide into carbon monoxide and the coupled synthesis of acetyl-CoA from the carbon monoxide produced. Exposure of CODH/ACS from Moorella thermoacetica to carbon monoxide gives rise to several infrared bands in the 2100-1900 cm(-1) spectral region that are attributed to the formation of metal-coordinated carbon monoxide species. Infrared bands attributable to M-CO are not detected in the as-isolated enzyme, suggesting that the enzyme does not contain intrinsic metal-coordinated CO ligands. A band detected at 1996 cm(-1) in the CO-flushed enzyme is assigned as arising from CO binding to a metal center in cluster A of the ACS subunit. The frequency of this band is most consistent with it arising from a terminally coordinated Ni(I) carbonyl. Multiple infrared bands at 2078, 2044, 1970, 1959, and 1901 cm(-1) are attributed to CO binding at cluster C of the CODH subunit. All infrared bands attributed to metal carbonyls decay in a time-dependent fashion as CO(2) appears in the solution. These observations are consistent with the enzyme-catalyzed oxidation of carbon monoxide until it is completely depleted from solution during the course of the experiments.     

The catalytic role of the copper ligand H172 of peptidylglycine alpha-hydroxylating monooxygenase: a kinetic study of the H172A mutant

An essential histidine ligand to the electron transfer copper (CuH) of peptidylglycine alpha-hydroxylating monooxygenase (PHMcc) was mutated to an alanine and found to retain copper binding and hydroxylase activity [Jaron, S., et al. (2002) Biochemistry 41, 13274-13282]. An extensive kinetic and deuterium isotope effect study finds this mutant to maintain full coupling of O2 consumed to product formed despite a 3 order-of-magnitude decrease in kcat and a 300-fold decrease in kcat/Km(O2). Unexpectedly, electron transfer is not rate-limiting in H172A. Rather, the increased kinetic isotope effect (KIE) on kcat of 3.27 +/- 0.39 suggests that C-H bond cleavage has become more rate-limiting, implicating a role for His172 that goes beyond that of a simple ligand to CuH. The mechanistic implications are discussed.     

Glutathione-mediated transfer of copper(I) into American lobster apohemocyanin.

Copper in the cytosol of the hepatopancreas of the American lobster, Homarus americanus, occurs as copper-metallothionein [Cu(I)-MT] and as a copper-glutathione complex [Cu(I)-GSH]. The latter can act in vitro as the source of Cu(I) in the reconstitution of lobster apohemocyanin, whereas Cu(I)-MT cannot. Here we report on the mechanism of the GSH-mediated reconstitution. Binding of Cu(I) to apohemocyanin was measured by its effect on the protein's fluorescence, by ultrafiltration experiments and size-exclusion HPLC. Reconstitution of CO and O2 binding was studied using the [Cu(I)...Cu(I)-CO] fluorescence of hemocyanin and its Cu-O2-Cu charge-transfer band as spectral probes. The hemocyanin oligomer has 1 (1.02 +/- 0.09) high-affinity (apparent Kdiss = 1.67 +/- 0.40 microM) external binding site for ionic Cu(I) per subunit. Binding of Cu(I) to this site is fast and reversible and is followed by a slow, irreversible incorporation of copper into the protein matrix. Movement of the first copper through the matrix to the active site is the rate-limiting step in the reconstitution process. Mononuclear copper sites, once formed, are rapidly converted into biologically active, binuclear copper sites. In accordance with this reaction sequence, the restoration of CO/O2 binding by hemocyanin is a first-order reaction with a half-time of 100 +/- 5 min at pH 6.0. Reconstitution is extremely pH-dependent and proceeds best at those pH values where the architecture of the copper pocket of hemocyanin is open as judged from its extremely low affinity for oxygen and its very fast oxygen dissociation rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Copper(I)-phenolate complexes as models of the reduced active site of galactose oxidase: synthesis, characterization, and O2 reactivity

The Cu(I)-phenolate complexes (1)LCu and (2)LCu and the Cu(I)-phenol complex [H(2)LCu(CNC(6)H(3)Me(2))]BArF(4) were prepared and structurally characterized by X-ray crystallography, where (1)L(-) and (2)L(-) are ligands comprised of a 2,4-di- tert-butylphenolate linked to 1-isopropyl-1,5-diazacyclooctane or 1,4-diisopropyl-1,4,7-triazacyclononane, respectively. The reduced galactose oxidase (GAO) structural models (1)LCu and (2)LCu were found to be highly reactive with O(2), and through combined stopped-flow kinetic and EPR, UV-vis, and resonance Raman spectroscopic studies of the oxygenation of (2)LCu at low temperature, new intermediates relevant to those postulated for the active site oxidation step of the GAO catalytic cycle were identified. The oxygenation was shown by kinetics experiments to proceed via initial binding of O(2) to yield a green, unusually thermodynamically stable 1:1 adduct, (2)LCu(O(2)). Symmetric (eta(2)) binding of a superoxo ligand was indicated by oxygen-isotope-sensitive features in resonance Raman spectra obtained in batch experiments; peaks at nu((16)O(2))=1120 cm(-1), nu((18)O(16)O)=1093 cm(-1), and nu((18)O(2))=1058 cm(-1) were assigned as O-O stretching vibrations. These data represent the first experimental evidence for such superoxide coordination in complexes of tetradentate tripodal ligands and provide new precedent for how O(2) may bind at the reduced GAO active site. The 1:1 Cu/O(2) adduct subsequently evolves into a metastable purple species that is only observable under conditions of substoichiometric O(2). The kinetics of formation of this transient species are second order overall (rate= k'(2)[(2)LCu(O(2))][(2)LCu]). It exhibits an absorption band with lambda(max)=565 nm (epsilon=17900 M(-1) cm(-1)) and multiple oxygen-isotope-sensitive nu(Cu-O) and nu(O-O) features in the respective regions 500-550 cm(-1) and 700-850 cm(-1) in Raman spectra, with excitation-wavelength-dependent intensities that correlate with the 565 nm absorption feature. On the basis of the combined data available, the presence of multiple isomeric peroxodicopper species in the transient purple solution is postulated.     

Copper(I) transfer into apo-stellacyanin using copper(I)-thiourea as a copper-thionein model.

The direct incorporation of Cu(I) from [Cu(I)(thiourea)3]Cl, a structural analogue of Cu-thionein, into apo-stellacyanin, was successful both aerobically and anaerobically. A characteristic c.d. band of Cu(I)-stellacyanin at 270 nm (0 = -12.5 X 10(3) degrees X cm2 X dmol-1) was seen. On oxidation with hexacyanoferrate(III) or by air, the correct Cu(II) binding into the active centre of this 'Type 1' Cu-protein was deduced from chiroptical measurements which were supported by e.p.r. data. Thus Cu-thiourea turned out to be an excellent Cu(I)-donor in aqueous systems for the complete reconstitution of mononuclear Blue copper proteins.     

Coordination mode and reactivity of copper(II) complexes with kasugamycin.

Protonation and Cu(II) coordination of kasugamycin were studied by potentiometry, UV-vis, CD, EPR, 13C NMR, and 1H NMR. Mononuclear complexes with stoichiometries ranging from CuHL to CuH(-1)L were found. The aminoamidine moiety provides the coordination site in the CuHL species. The additional axial coordination of the amino nitrogen of the aminosugar ring is present in CuL. Finally, the CuH(-1)L complex is formed as a result of a deprotonation and coordination of the hydroxyl group of the inositol ring. The non-planar arrangement of the chelate rings results in the relative stabilization of a Cu(I) species. As a consequence, Cu(I) and superoxide radicals are involved in the redox mechanism of H(2)O(2) activation by the Cu(II) complex of kasugamycin.     

Spectroscopic characterization of mononitrosyl complexes in heme--nonheme diiron centers within the myoglobin scaffold (Fe(B)Mbs): relevance to denitrifying NO reductase

Denitrifying NO reductases are evolutionarily related to the superfamily of heme--copper terminal oxidases. These transmembrane protein complexes utilize a heme-nonheme diiron center to reduce two NO molecules to N(2)O. To understand this reaction, the diiron site has been modeled using sperm whale myoglobin as a scaffold and mutating distal residues Leu-29 and Phe-43 to histidines and Val-68 to a glutamic acid to create a nonheme Fe(B) site. The impact of incorporation of metal ions at this engineered site on the reaction of the ferrous heme with one NO was examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. UV--vis absorption and resonance Raman spectra demonstrate that the first NO molecule binds to the ferrous heme, but while the apoproteins and Cu(I)- or Zn(II)-loaded proteins show characteristic EPR signatures of S = 1/2 six-coordinate heme {FeNO}(7) species that can be observed at liquid nitrogen temperature, the Fe(II)-loaded proteins are EPR silent at ≥30 K. Vibrational modes from the heme [Fe-N-O] unit are identified in the RR and FTIR spectra using (15)NO and (15)N(18)O. The apo and Cu(I)-bound proteins exhibit ν(FeNO) and ν(NO) that are only marginally distinct from those reported for native myoglobin. However, binding of Fe(II) at the Fe(B) site shifts the heme ν(FeNO) by 17 cm(-1) and the ν(NO) by -50 cm(-1) to 1549 cm(-1). This low ν(NO) is without precedent for a six-coordinate heme {FeNO}(7) species and suggests that the NO group adopts a strong nitroxyl character stabilized by electrostatic interaction with the nearby nonheme Fe(II). Detection of a similarly low ν(NO) in the Zn(II)-loaded protein supports this interpretation.     

Structure and coordination of CuB in the Acidianus ambivalens aa 3 quinol oxidase heme–copper center

The coordination environment of the Cu_B center of the quinol oxidase from Acidianus ambivalens, a type B heme–copper oxygen reductase, was investigated by Fourier transform (FT) IR and extended X-ray absorption fine structure (EXAFS) spectroscopy. The comparative structural chemistry of dinuclear Fe–Cu sites of the different types of oxygen reductases is of great interest. Fully reduced A. ambivalens quinol oxidase binds CO at the heme a ₃ center, with ν(CO)=1,973 cm⁻¹. On photolysis, the CO migrated to the Cu_B center, forming a Cu_B^I–CO complex with ν(CO)=2,047 cm⁻¹. Raising the temperature of the samples to 25°C did not result in a total loss of signal in the FTIR difference spectrum although the intensity of these signals was reduced sevenfold. This observation is consistent with a large energy barrier against the geminate rebinding of CO to the heme iron from Cu_B, a restricted limited access at the active-site pocket for a second binding, and a kinetically stable Cu_B–CO complex in A. ambivalens aa ₃. The Cu_B center was probed in a number of different states using EXAFS spectroscopy. The oxidized state was best simulated by three histidines and a solvent O scatterer. On reduction, the site became three-coordinate, but in contrast to the bo ₃ enzyme, there was no evidence for heterogeneity of binding of the coordinated histidines. The Cu_B centers in both the oxidized and the reduced enzymes also appeared to contain substoichiometric amounts (0.2 mol equiv) of nonlabile chloride ion. EXAFS data of the reduced carbonylated enzyme showed no difference between dark and photolyzed forms. The spectra could be well fit by 2.5 imidazoles, 0.5 Cl⁻ and 0.5 CO ligands. This arrangement of scatterers would be consistent with about half the sites remaining as unligated Cu(his)₃ and half being converted to Cu(his)₂Cl⁻CO, a 50/50 ratio of Cu(his)₂Cl⁻ and Cu(his)₃CO, or some combination of these formulations. Electronic Supplementary Material Supplementary material is available for this article at .     

FTIR studies of the CO and cyanide adducts of fully reduced bovine cytochrome c oxidase

Photolysis spectra of the CO and cyanide adducts of reduced bovine cytochrome c oxidase have been studied by FTIR difference spectroscopy. Bound CO is predominantly in a single 1963 cm(-1) form whereas cyanide is bound in at least two forms (2058/2045 cm(-1)). These forms are pH-independent between pH 6.5 and 8.5, indicating that there is no titratable protonatable group that influences significantly their binding in this pH range. Photolysis spectra of the cyanide adduct have a positive band around 2090 cm(-1) in H(2)O due at least in part to free HCN and at 1880 cm(-1) in D(2)O due to free DCN. The frequency of the positive band around 2090 cm(-1), and its persistence in D(2)O media, raises the possibility that a transient cyanide-Cu(B) adduct also contributes to this signal, equivalent to the CO-Cu(B) species that is formed when CO is photolyzed. Photolysis produces changes throughout the 1000-1800 cm(-1) region. Reduced minus (reduced + CO) photolysis spectra in H(2)O exhibit a pH-independent and symmetrical peak/trough at 1749/1741 cm(-1). A related feature in homologous oxidases has been suggested to arise from a conserved glutamic acid. However, only around one-third of the feature is shifted to lower frequencies by incubation in D(2)O media, and an additional fraction is shifted if catalytic turnover occurs in D(2)O. Reduced minus (reduced + cyanide) photolysis spectra exhibit multiple features in H(2)O in this region with peaks at 1752, 1725, and 1708 cm(-1) and troughs at 1740, 1715, and 1698 cm(-1). Again, only a part of these features shift in D(2)O, even with catalytic turnover. A variety of additional H/D-sensitive features in the 1700-1000 cm(-1) region of the spectra can be discerned, one of which in cyanide photolysis spectra is tentatively assigned to a conserved tyrosine, Y244. Data are discussed in relation to the structure of the binuclear center and protonatable groups in its vicinity.     

Fourier transform infrared (FTIR) and step-scan time-resolved FTIR spectroscopies reveal a unique active site in cytochrome caa3 oxidase from Thermus thermophilus

Fourier transform infrared (FTIR) and step-scan time-resolved FTIR difference spectra are reported for the [carbonmonoxy]cytochrome caa(3) from Thermus thermophilus. A major C-O mode of heme a(3) at 1958 cm(-1) and two minor modes at 1967 and 1975 cm(-1) (7:1:1) have been identified at room temperature and remained unchanged in H(2)O/D(2)O exchange. The observed C-O frequencies are 10 cm(-1) higher than those obtained previously at 21 K (Einarsdóttir, O., Killough, P. M., Fee, J. A., and Woodruff, W. H. (1989) J. Biol. Chem. 264, 2405-2408). The time-resolved FTIR data indicate that the transient Cu(B)(1+)-CO complex is formed at room temperature as revealed by the CO stretching mode at 2062 cm(-1). Therefore, the caa(3) enzyme is the only documented member of the heme-copper superfamily whose binuclear center consists of an a(3)-type heme of a beta-form and a Cu(B) atom of an alpha-form. These results illustrate that the properties of the binuclear center in other oxidases resulting in the alpha-form are not required for enzymatic activity. Dissociation of the transient Cu(B)(1+)-CO complex is biphasic. The rate of decay is 2.3 x 10(4) s(-1) (fast phase, 35%) and 36.3 s(-1) (slow phase, 65%). The observed rate of rebinding to heme a(3) is 34.1 s(-1). The implications of these results with respect to the molecular motions that are general to the photodynamics of the binuclear center in heme-copper oxidases are discussed.     

FTIR studies of internal proton transfer reactions linked to inter-heme electron transfer in bovine cytochrome c oxidase

FTIR difference spectroscopy is used to reveal changes in the internal structure and amino acid protonation states of bovine cytochrome c oxidase (CcO) that occur upon photolysis of the CO adduct of the two-electron reduced (mixed valence, MV) and four-electron reduced (fully reduced, FR) forms of the enzyme. FTIR difference spectra were obtained in D(2)O (pH 6-9.3) between the MV-CO adduct (heme a(3) and Cu(B) reduced; heme a and Cu(A) oxidized) and a photostationary state in which the MV-CO enzyme is photodissociated under constant illumination. In the photostationary state, part of the enzyme population has heme a(3) oxidized and heme a reduced. In MV-CO, the frequency of the stretch mode of CO bound to ferrous heme a(3) decreases from 1965.3 cm(-1) at pH* 相似文献