Bohr-effect and pH-dependence of electron spin resonance spectra of a cobalt-substituted monomeric insect haemoglobin

The monomeric haemoglobin IV from Chironomus thummi thummi (CTT IV) exhibits an alkaline Bohr-effect and therefore it is an allosteric protein. By substitution of the haem iron for cobalt the O2 half-saturation pressure, measured at 25 degrees C, increases 250-fold. The Bohr-effect is not affected by the replacement of the central atom. The parameters of the Bohr-effect of cobalt CTT IV for 25 degrees C are: inflection point of the Bohr-effect curve at pH 7.1, number of Bohr protons -- deltalog p1/2 (O2)/deltapH = 0.36 mol H+/mol O2 and amplitude of the Bohr-effect curve deltalogp1/2 (O2) = 0.84. The substitution of protoporphyrin for mesoporphyrin causes a 10 nm blue-shift of the visible absorption maxima in both, the native and the cobalt-substituted forms of CTT IV. Furthermore, the replacement of vinyl groups by ethyl groups at position 2 and 4 of the porphyrin system leads to an increase of O2 affinities at 25 degrees C which follows the order: proto less than meso less than deutero for iron and cobalt CTT IV, respectively. Again, the Bohr-effect is not affected by the replacement of protoporphyrin for mesoporphyrin or deuteroporphyrin. The electron spin resonance (ESR) spectra of both, deoxy cobalt proto- and deoxy cobalt meso-CTT IV, are independent of pH. The stronger electron-withdrawing effect by protoporphyrin is reflected by the decrease of the cobalt hyperfine constants coinciding with gparallel = 2.035 and by the low-field shift of gparallel. The ESR spectra of oxy cobalt proto- and oxy cobalt meso-CTT IV are dependent of pH. The cobalt hyperfine constants coinciding with gparallel - 2.078 increase during transition from low to high pH. The pH-induced ESR spectral changes correlate with the alkaline Bohr-effect. Therefore, the two O2 affinity states can be assigned to the low-pH and high-pH ESR spectral species. The low-pH form (low-affinity state) is characterized by a smaller, the high-pH form (high-affinity state) by a larger cobalt hyperfine constant in gparallel. The correlation of the cobalt hyperfine constants of the oxy forms with the O2 affinities is discussed for several monomeric haemoglobins. The Co-O-O bond angle in cobalt oxy CTT IV is characterized by an ozonoid type of binding geometry and varies little during the pH-induced conformation transition. Due to the lack of the distal histidine in CTT IV no additional interaction via hydrogen-bonding with dioxygen is possible; this is reflected by the cobalt hyperfine constants.     

Biochemical characterization and solution structure of nitrous oxide reductase from Alcaligenes xylosoxidans (NCIMB 11015).

Nitrous oxide reductase (N2OR) is the terminal enzyme involved in denitrification by microbes. No three-dimensional structural information has been published for this enzyme. We have isolated and characterised N2OR from Alcaligenes xylosoxidans (AxN2OR) as a homodimer of M(r) 134,000 containing seven to eight copper atoms per dimer. Comparison of sequence and compositional data with other N2ORs suggests that AxN2OR is typical and can be expected to have similar domain folding and subunit structure to other members of this family of enzymes. We present synchrotron X-ray-scattering data, analysed using a model-independent method for shape restoration, which gave a approximately 20 A resolution structure of the enzyme in solution, providing a glimpse of the structure of any N2OR and shedding light on the molecular architecture of the molecule. The specific activity of AxN2OR was approximately 6 mumol of N2O reduced.min-1. (mg of protein)-1; N2OR activity showed both base and temperature activation. The visible spectrum exhibited an absorption maximum at 550 nm with a shoulder at 635 nm. On oxidation with K3Fe(CN)6, the absorption maximum shifted to 540 nm and a new shoulder at 480 nm appeared. Reduction under anaerobic conditions resulted in the formation of an inactive blue form of the enzyme with a broad absorption maximum at 650 nm. As isolated, the enzyme shows an almost featureless EPR spectrum, which changes on oxidation to give an almost completely resolved seven-line hyperfine signal in the gII region, g = 2.18, with AII = 40 G, consistent with the enzyme being partially reduced as isolated. Both the optical and EPR spectra of the oxidized enzyme are characteristic of the presence of a CuA centre.     

Electron transfer after flash photolysis of mixed-valence carboxycytochrome c oxidase

The light-induced difference spectra of the fully reduced (a2+ a23+-CO) complex and the mixed-valence carboxycytochrome c oxidase (a3+ a23+-CO) during steady-state illumination and after flash photolysis showed marked differences. The differences appear to be due to electron transfer between the redox centres in the enzyme. The product of the absorbance coefficient and the quantum yield was found to be equal in both enzyme species, both when determined from the rates of photolysis and from the values of the dissociation constants of the cytochrome a23+-CO complex. This would confirm that the spectral properties of cytochrome a3 are not affected by the redox state of cytochrome a and CuA. When the absorbance changes after photolysis of cytochrome a23+-CO with a laser flash were followed on a time scale from 1 mus to 1 s in the fully reduced carboxycytochrome c oxidase, only the CO recombination reaction was observed. However, in the mixed-valence enzyme an additional fast absorbance change (k = 7 X 10(3) s-1) was detected. The kinetic difference spectrum of this fast change showed a peak at 415 nm and a trough at 445 nm, corresponding to oxidation of cytochrome a3. Concomitantly, a decrease of the 830 nm band was observed due to reduction of CuA. This demonstrates that in the partially reduced enzyme a pathway is present between CuA and the cytochrome a3-CuB pair, via which electrons are transferred rapidly.     

Low temperature phototransformations of protochlorophyll(ide) in etiolated leaves

By methods of difference and derivative spectroscopy it was shown that in etiolated leaves at 77 K three photoreactions of P650 protochlorophyllide take place which differ in their rates and positions of spectral maxima of the intermediates formed in the process: P650R668, P650R688, and P650R697. With an increase of temperature up to 233 K, in the dark, R688 and R697 are transformed into the known chlorophyllide forms C695/684 and C684/676, while R668 disappears with formation of a shorter wavelength form of protochlorophyllide with an absorption maximum at 643–644 nm.Along with these reactions, at 77 K phototransformations of the long-wave protochlorophyllide forms with absorption maxima at 658–711 nm into the main short-wave forms of protochlorophyllide are observed. At 233 K in the dark this reaction is partially reversible. This process may be interpreted as a reversible photodisaggregation of the pigment in vivo.The mechanism of P650 reactions and their role in the process of chlorophyll photobiosynthesis are discussed.Abbreviations P650 protochlorophyll(ide) with absorption maximum at 650 nm - C697/684 chlorophyllide with fluorescence maximum at 695 nm and absorption maximum at 684 nm - R697 intermediate with absorption maximum at 697 nm     

The optical properties of CuA in bovine cytochrome c oxidase determined by low-temperature magnetic-circular-dichroism spectroscopy.

The visible-near-i.r.-region m.c.d. (magnetic-circular-dichroism) spectrum recorded at low temperature in the range 450-900 nm is reported for oxidized resting mammalian cytochrome c oxidase. M.c.d. magnetization curves determined at different wavelengths reveal the presence of two paramagnetic species. Curves at 576, 613 and 640 nm fit well to those expected for an x,y-polarized haem transition with g values of 3.03, 2.21 and 1.45, i.e. cytochrome a3+. The m.c.d. features at 515, 785 and 817 nm magnetize as a S = 1/2 paramagnet with average g values close to 2, and simulated m.c.d. magnetization curves obtained by using the observed g values of CuA2+, i.e. 2.18, 2.03 and 1.99, fit well to the experimental observations. The form of the m.c.d. magnetization curve at 466 nm is curious, but it can be explained if CuA2+ and cytochrome a3+ contribute with oppositely signed bands at this wavelength. By comparing the m.c.d. spectrum of the enzyme with that of extracted haem a-bisimidazole complex it has been possible to deconvolute the m.c.d. spectrum of CuA2+, which shows transitions throughout the spectral region from 450 to 950 nm. The m.c.d.-spectral properties of CuA2+ were compared with those of a well-defined type I blue copper centre in azurin isolated from Pseudomonas aeruginosa. The absolute intensities of the m.c.d. signals at equal fields and temperatures for CuA2+ are 10-20-fold greater than those for azurin. The optical spectrum of CuA2+ strongly suggests an assignment as a d9 ion rather than Cu(I) bound to a thiyl radical.     

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.     

Cytochrome c peroxidase. Interconversion of chemically and enzymatically reactive and unreactive forms of the ferric protein.

Ferric yeast cytochrome c peroxidase in the presence of different anions may assume a number of forms which differ in optical spectra and chemical properties. In solutions whose only anion is acetate, two spectral forms are present together in an equilibrium. Each of these spectral species is believed to bear bound acetate anion. A form characterized by an intense absorption maximum at 620 nm is unreactive enzymatically and does not react with hydrogen peroxide or with dithionite. A form characterized by a less intense absorption near 645 nm is enzymatically and chemically reactive. Increasing temperature and increasing pH displace the equilibrium toward the 645 nm form. Increasing cytochrome c peroxidase concentration favors the 620 nm form. In kinetic experiments in which the 645 nm form is removed by rapid reaction with H2O2 or dithionite, the 620 nm form is converted in a first order reaction (k = 0.36 s-1, 15 degrees C) to the 645 nm form. In solutions whose sole anion is phosphate a 645 nm form is the only demonstrable spectral species. The enzymatic activity and rates of chemical reaction of 645 nm spectral forms occurring in acetate and in phosphate buffers are the same.     

An engineered CuA Amicyanin capable of intermolecular electron transfer reactions

The type I copper center of amicyanin was replaced with a binuclear CuA center. To create this model CuA protein, a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Paracoccus denitrificans amicyanin was replaced with the corresponding portion of sequence that provides five ligands for the CuA center of cytochrome c oxidase from P. denitrificans. UV-visible and electron paramagnetic resonance spectroscopy confirm that the engineered protein as isolated possesses the mixed-valence Cu1.5Cu1.5 (purple) CuA center. Comparison of the spectroscopic properties of this CuA amicyanin with those of the CuA centers of other natural and engineered CuA proteins suggests that the spectroscopic features may be dictated more by the protein host than the sequence of the CuA loop. Novel reactions for a simple CuA model protein are also described. In contrast to other natural and engineered CuA proteins, the fully reduced CuA amicyanin may be reoxidized by molecular oxygen to the mixed-valence state. It is also shown that CuA amicyanin can serve as an electron donor and an electron acceptor for other redox proteins. The mixed-valence form accepts electrons from cytochromes c-551i and c-550 from P. denitrificans. The fully reduced form donates electrons to native and P94F amicyanin. The function as either an electron donor or acceptor is consistent with the measured redox potential of CuA amicyanin of +273 mV. These data indicate that this CuA amicyanin will be a particularly useful model protein for structure-function studies of reactivity and the electron transfer properties of the CuA redox center.     

An electron nuclear double resonance investigation of redox-induced electronic structural change at CuA2+ in cytochrome c oxidase

We measured an electronic change at cysteine ligand(s) of the CuA2+ center brought on by reduction of other metal centers within cytochrome c oxidase, notably cytochrome a. This change specifically manifested itself as a modification in magnetic hyperfine coupling to the beta-protons of the beta-carbons adjacent to the cysteine sulfur in the CuA2+ coordination sphere. The electron nuclear double resonance ENDOR signals of these beta-protons had previously been assigned through study of selectively deuterated yeast oxidase. In the present study the ENDOR signals of the CuA2+ center were compared from the following forms of oxidase: resting (a3+.CuA2+.a3+3.CuB2+); mixed valence, 2-electron-reduced CO-ligated oxidase (a3+.CuA2+.a2+3CO.CuB+), and a more completely reduced mixed-valence CO-ligated oxidase. In agreement with previous studies on 3-electron-reduced oxidase, the latter more completely reduced oxidase showed cytochrome a preferentially reduced with respect to CuA, implying that the majority of paramagnetic CuA2+ centers had reduced cytochrome a partners. The ENDOR-resolved splitting of the beta-proton hyperfine features substantially decreased in going from the first two more oxidized forms to the more fully reduced latter form. Thus, the electronic structure of the CuA2+ center specifically monitored by hyperfine couplings to cysteine protons changed in response to a reductive event elsewhere in the protein. This structural change may correlate with the anticooperative redox interaction recently reported between cytochrome a and CuA.     

Redox-linked structural changes associated with the formation of a catalytically competent form of the diheme cytochrome c peroxidase from Pseudomonas aeruginosa

A recombinant form of the prototypic diheme bacterial cytochrome c peroxidase (BCCP) from Pseudomonas aeruginosa (PsaCCP) has been expressed in Escherichia coli and purified to homogeneity. This material was used to carry out the first integrated biochemical, spectroscopic and structural investigation of the factors leading to reductive activation of this class of enzymes. A single, tightly bound, Ca2+ ion (K = 3 x 10(10) M-1) found at the domain interface of both the fully oxidized and mixed-valence forms of the enzyme is absolutely required for catalytic activity. Reduction of the electron-transferring (high-potential) heme in the presence of Ca2+ ions triggers substantial structural rearrangements around the active-site (low-potential) heme to allow substrate binding and catalysis. The enzyme also forms a mixed-valence state in the absence of Ca2+ ions, but a combination of electronic absorption, and EPR spectroscopies suggests that under these circumstances the low potential heme remains six-coordinate, unable to bind substrate and therefore catalytically inactive. Our observations strongly suggest that the two mixed-valence forms of native PsaCCP reported previously by Foote and colleagues (Foote, N., Peterson, J., Gadsby, P., Greenwood, C., and Thomson, A. (1985) Biochem. J. 230, 227-237) correspond to the Ca2+-loaded and -depleted forms of the enzyme.     

Structural basis of electron transfer modulation in the purple CuA center

The X-ray structure of an engineered purple CuA center in azurin from Pseudomonas aeruginosa has been determined and refined at 1.65 A resolution. Two independent purple CuA azurin molecules are in the asymmetric unit of a new P21 crystal, and they have nearly identical conformations (rmsd of 0.27 A for backbone atoms). The purple CuA azurin was produced by the loop-engineering strategy, and the resulting overall structure is unperturbed. The insertion of a slightly larger Cu-binding loop into azurin causes the two structural domains of azurin to move away from each other. The high-resolution structure reveals the detailed environment of the delocalized mixed-valence [Cu(1.5).Cu(1.5)] binuclear purple CuA center, which serves as a useful reference model for other native proteins, and provides a firm basis for understanding results from spectroscopic and functional studies of this class of copper center in biology. The two independent Cu-Cu distances of 2.42 and 2.35 A (with respective concomitant adjustments of ligand-Cu distances) are consistent with that (2.39 A) obtained from X-ray absorption spectroscopy with the same molecule, and are among the shortest Cu-Cu bonds observed to date in proteins or inorganic complexes. A comparison of the purple CuA azurin structure with those of other CuA centers reveals an important relationship between the angular position of the two His imidazole rings with respect to the Cu2S2(Cys) core plane and the distance between the Cu and the axial ligand. This relationship strongly suggests that the fine structural variation of different CuA centers can be correlated with the angular positions of the two histidine rings because, from these positions, one can predict the relative axial ligand interactions, which are responsible for modulating the Cu-Cu distance and the electron transfer properties of the CuA centers.     

Intrinsic fluorescence of B and Z forms of poly d(G-m5C).poly d(G-m5C), a synthetic double-stranded DNA: spectra and lifetimes by the maximum entropy method.

A study has been made of the fluorescence of poly d(G-m5C).poly d(G-m5C), a synthetic double-stranded DNA, in buffered neutral aqueous solution at room temperature, excited by synchrotron radiation at 280 nm and 250 nm and by a frequency-doubled pulse dye laser at 290 nm. Exciting at 280 nm, the B form shows a uni-modal UV spectrum with lambdaf(max) approximately 340 nm. The Z form has in addition a visible emission lambdaf(max) at 450 nm. The spectral positions remain unchanged on exciting at 250 nm but the relative intensities change considerably. Decay profiles have been obtained at 360 nm and 450 nm for both the B and Z forms and have been analyzed by fitting to a pseudo-continuous distribution of 100 (and occasionally 200) exponentials, ranging from 10 ps to 20 ns, by optimizing the 'entropy' of the signal (the method of maximum entropy). We find the mean lifetimes for both wavelengths of emission and for both structural forms fall into three well-separated regions in the ranges indicated tau1 approximately 0.04-0.21 ns, tau2 approximately 0.9-1.26 ns, and tau3 approximately 5.1-6.5 ns. The UV emission, from its spectral position and half-width, correlates with monomeric emission from m5C (and from C for poly d(G-C)). However the lifetime tau1 is approximately 2 orders of magnitude longer than the monomers and points to an involvement of protonated guanosine (GH+, tauf approximately 200 ps) in the overall absorption/emission sequence. In the UV the tau3 emission is predominant, with fractional time-integrated emission approximately 86% for B DNA and approximately 64% for Z. We suggest it results from exciton (stacked) absorption followed by dissociative emission. For Z DNA the visible (450 nm) emission is dominated by a tau3 species (approximately 91%) with a lifetime of 6.5 ns and we suggest it represents a hetero-excimer emission consequent upon absorption by the strongly overlapped base-stacking, which differs from that in B DNA. The weak emission corresponding to tau2 is made more apparent by scanned gated detection of the emission from laser excitation (290 nm) of single-crystal d(m5C-G)3. A central role is attributed to the tight stacking of the bases in the Z form which correlates with enhanced hypochromism at 250 nm vs. 280 nm and with the reversal of the fluorescence intensity ratios UV-visible between these wavelengths.     

Structural investigations of the CuA centre of nitrous oxide reductase from Pseudomonas stutzeri by site-directed mutagenesis and X-ray absorption spectroscopy.

Nitrous oxide reductase is the terminal component of a respiratory chain that utilizes N2O in lieu of oxygen. It is a homodimer carrying in each subunit the electron transfer site, CuA, and the substrate-reducing catalytic centre, CuZ. Spectroscopic data have provided robust evidence for CuA as a binuclear, mixed-valence metal site. To provide further structural information on the CuA centre of N2O reductase, site directed mutagenesis and Cu K-edge X-ray absorption spectroscopic investigation have been undertaken. Candidate amino acids as ligands for the CuA centre of the enzyme from Pseudomonas stutzeri ATCC14405 were substituted by evolutionary conserved residues or amino acids similar to the wild-type residues. The mutations identified the amino acids His583, Cys618, Cys622 and Met629 as ligands of Cu1, and Cys618, Cys622 and His626 as the minimal set of ligands for Cu2 of the CuA centre. Other amino acid substitutions indicated His494 as a likely ligand of CuZ, and an indirect role for Asp580, compatible with a docking function for the electron donor. Cu binding and spectroscopic properties of recombinant N2O reductase proteins point at intersubunit or interdomain interaction of CuA and CuZ. Cu K-edge X-ray absorption spectra have been recorded to investigate the local environment of the Cu centres in N2O reductase. Cu K-edge Extended X-ray Absorption Fine Structure (EXAFS) for binuclear Cu chemical systems show clear evidence for Cu backscattering at approximately 2.5 A. The Cu K-edge EXAFS of the CuA centre of N2O reductase is very similar to that of the CuA centre of cytochrome c oxidase and the optimum simulation of the experimental data involves backscattering from a histidine group with Cu-N of 1.92 A, two sulfur atoms at 2.24 A and a Cu atom at 2. 43 A, and allows for the presence of a further light atom (oxygen or nitrogen) at 2.05 A. The interpretation of the CuA EXAFS is in line with ligands assigned by site-directed mutagenesis. By a difference spectrum approach, using the Cu K-edge EXAFS of the holoenzyme and that of the CuA-only form, histidine was identified as a major contributor to the backscattering. A structural model for the CuA centre of N2O reductase has been generated on the basis of the atomic coordinates for the homologous domain of cytochrome c oxidase and incorporating our current results and previous spectroscopic data.     

Origin of the 701-nm fluorescence emission of the Lhca2 subunit of higher plant photosystem I

Photosystem I of higher plants is characterized by red-shifted spectral forms deriving from chlorophyll chromophores. Each of the four Lhca1 to -4 subunits exhibits a specific fluorescence emission spectrum, peaking at 688, 701, 725, and 733 nm, respectively. Recent analysis revealed the role of chlorophyll-chlorophyll interactions of the red forms in Lhca3 and Lhca4, whereas the basis for the fluorescence emission at 701 nm in Lhca2 is not yet clear. We report a detailed characterization of the Lhca2 subunit using molecular biology, biochemistry, and spectroscopy and show that the 701-nm emission form originates from a broad absorption band at 690 nm. Spectroscopy on recombinant mutant proteins assesses that this band represents the low energy form of an excitonic interaction involving two chlorophyll a molecules bound to sites A5 and B5, the same protein domains previously identified for Lhca3 and Lhca4. The resulting emission is, however, substantially shifted to higher energies. These results are discussed on the basis of the structural information that recently became available from x-ray crystallography (Ben Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630-635). We suggest that, within the Lhca subfamily, spectroscopic properties of chromophores are modulated by the strength of the excitonic coupling between the chromophores A5 and B5, thus yielding fluorescence emission spanning a large wavelength interval. It is concluded that the interchromophore distance rather than the transition energy of the individual chromophores or the orientation of transition vectors represents the critical factor in determining the excitonic coupling in Lhca pigment-protein complexes.     

Near infrared spectral changes of cytochrome aa3 during potentiometric titrations.

Singular value decomposition (SVD) was used to deconvolute the spectral changes occurring in the near infrared region during potentiometric titrations of cytochrome aa3. Overall oxidized minus reduced difference spectra revealed a broad absorbance feature centered near 830 nm with an apparent Em near 250 mV. However, SVD did not isolate any spectral species with an absorbance centered near 830 nm. It was found that the spectral changes occurring in the wavelength region from 650 to 950 nm were associated mainly with cytochromes a and a3. It was concluded that the absorbance at 830 nm should not be used as an independent measure of the concentration of CuA in cytochrome aa3.     

Probing protonation/deprotonation of tyrosine residues in cytochrome ba3 oxidase from Thermus thermophilus by time-resolved step-scan Fourier transform infrared spectroscopy

Elucidating the properties of the heme Fe-Cu(B) binuclear center and the dynamics of the protein response in cytochrome c oxidase is crucial to understanding not only the dioxygen activation and bond cleavage by the enzyme but also the events related to the release of the produced water molecules. The time-resolved step-scan FTIR difference spectra show the ν(7a)(CO) of the protonated form of Tyr residues at 1247 cm(-1) and that of the deprotonated form at 1301 cm(-1). By monitoring the intensity changes of the 1247 and 1301 cm(-1) modes as a function of pH, we measured a pK(a) of 7.8 for the observed tyrosine. The FTIR spectral changes associated with the tyrosine do not belong to Tyr-237 but are attributed to the highly conserved in heme-copper oxidases Tyr-136 and/or Tyr-133 residue (Koutsoupakis, K., Stavrakis, S., Pinakoulaki, E., Soulimane, T., and Varotsis, C. (2002) J. Biol. Chem. 277, 32860-32866). The oxygenation of CO by the mixed-valence form of the enzyme revealed the formation of the ～607 nm P (Fe(IV)=O) species in the pH 6-9 range and the return to the oxidized form without the formation of the 580 nm F form. The data indicate that Tyr-237 is not involved in the proton transfer pathway in the oxygenation of CO by the mixed-valence form of the enzyme. The implication of these results with respect to the role of Tyr-136 and Tyr-133 in proton transfer/gating along with heme a(3) ring D propionate-H(2)O-ring A propionate-Asp-372 site to the exit/output proton channel (H(2)O pool) is discussed.     

Picosecond time-resolved absorption and fluorescence dynamics in the artificial bacteriorhodopsin pigment BR6.11.

The picosecond molecular dynamics in an artificial bacteriorhodopsin (BR) pigment containing a structurally modified all-trans retinal chromphore with a six-membered ring bridging the C11=C12-C13 positions (BR6.11) are measured by picosecond transient absorption and picosecond time-resolved fluorescence spectroscopy. Time-dependent intensity and spectral changes in absorption in the 570-650-nm region are monitored for delays as long as 5 ns after the 7-ps, 573-nm excitation of BR6.11. Two intermediates, J6.11 and K6.11/1, both with enhanced absorption to the red (> 600 nm) of the BR6.11 spectrum are observed within approximately 50 ps. The J6.11 intermediate decays with a time constant of 12 +/- 3 ps to form K6.11/1. The K6.11/1 intermediate decays with an approximately 100-ps time constant to form a third intermediate, K6.11/2, which is observed through diminished 650-nm absorption (relative to that of K6.11/1). No other transient absorption changes are found during the remainder of the initial 5-ns period of the BR6.11 photoreaction. Fluorescence in the 650-900-nm region is observed from BR6.11, K6.11/1, and K6.11/2, but no emission assignable to J6.11 is found. The BR6.11 fluroescence spectrum has a approximately 725-nm maximum which is blue-shifted by approximately 15 nm relative to that of native BR-570 and is 4.2 +/- 1.5 times larger in intensity (same sample optical density). No differences in the profile of the fluorescence spectra of BR6.11 and the intermediates K6.11/1 and K6.11/2 are observed. Following ground-state depletion of the BR6.11 population, the time-resolved fluroescence intensity monitored at 725 nm increases with two time constants, 12 +/- 3 and approximately 100 ps, both of which correlate well with changes in the picosecond transient absorption data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selenomethionine-substituted Thermus thermophilus cytochrome ba3: characterization of the CuA site by Se and Cu K-EXAFS.

We have designed a gene that encodes a polypeptide corresponding to amino acids 44-168 of the Thermus thermophilus cytochrome ba3 subunit II [Keightley et al. (1995) J. Biol. Chem. 270, 20345-20358]. The resulting ba3-CuAt10 protein separated into two fractions (A and B) during cation exchange chromatography which were demonstrated to differ only by N-terminal acetylation in fraction A. When the gene was expressed in an Escherichia coli strain that is auxotrophic for methionine and grown in the presence of selenomethionine (Se(Met)), the single methionine of the CuAt10 protein was quantitatively replaced with Se(Met). Native (S(Met)) and Se(Met)-substituted proteins were characterized by electrospray mass, optical absorption, and EPR spectroscopies and by electrochemical analysis; they were found to have substantially identical properties. The Se(Met)-containing protein was further characterized by Se and Cu K-EXAFS which revealed Cu-Se bond lengths of 2.55 A in the mixed-valence form and 2.52 A in the fully reduced form of CuA. Further analysis of the Se- and Cu-EXAFS spectra yielded the Se-S(thiolate) distances and thereby information on the Se-Cu-Cu and Se-Cu-S(thiolate) angles. An expanded EXAFS structural model is presented.     

7-Amino-actinomycin D as a cytochemical probe. I. Spectral properties.

The optical absorption and fluorescence characteristics of 7-animo-actinomycin D were determined to evaluate its potential as a fluorescent cytochemical probe. At pH 7.0, the absorption maximum and fluorescence excitation maximum are both at 503 nm; the fluorescence emission is at 675 nm. When this compound forms complexes with DNA in solution, the absorption and fluorescence excitation maxima shift to 543 nm and the fluorescence emission shifts to 655 nm. The fluorescence quantum yield is 0.016 for 7-amino-actinomycin D free in solution and 0.01-0.02 for complexes with native DNA. The 7-amino-actinomycin D also exhibits fluorescence shifts characteristic of binding when put into solution with poly(dG-dC) poly(dG-dC), but not with poly(dI-dC) poly(dI-dC). The spectral characteristics are the same at pH 7.0 whether the solvent is 0.01 M PO4 with 0.0001 M EDTA or Earle's salts with 0.025 M N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid.     

Purification, characterization, and preliminary crystallographic study of copper-containing nitrous oxide reductase from Pseudomonas nautica 617

The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named Cu(A) and Cu(Z). Cu(Z) could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)=2.015, A(x)=1.5 mT, g(y)=2.071, A(y)=2 mT, g(z)=2.138, A(z)=7 mT) and a strong absorption at approximately 640 nm. Cu(A) can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x)=g(y)= 2.021, A(x) = A(y)=0 mT, g(z) = 2.178, A(z)= 4 mT) and absorption bands at 480, 540, and approximately 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the Cu(A) center. In form A, Cu(A) is predominantly oxidized (S = (1)/(2), Cu(1.5+)-Cu(1.5+)), while in form B it is mostly in the one-electron reduced state (S = 0, Cu(1+)-Cu(1+)). In both forms, Cu(Z) remains reduced (S = 1/2). Complete crystallographic data at 2.4 A indicate that Cu(A) is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu(Z) is a novel tetracopper cluster [Brown, K., et al. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family.