Biomedical subjects
Publications and source records attributed to G M Clore.
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The glycosidic bond torsion angles and the conformations of the ribose of Mg2+ATP, Mg2+ADP and Mg2+AdoPP[NH]P (magnesium adenosine 5'-[beta, gamma-imido]triphosphate) bound to Ca2+ATPase, both native and modified with fluorescein isothiocyanate (FITC), in intact sarcoplasmic reticulum have been determined by the measurement of proton-proton transferred nuclear Overhauser enhancements by 1H-NMR spectroscopy. This method shows clearly the existence of a low-affinity ATP binding site after modification of the high-affinity site with FITC. For all three nucleotides bound to both the high-affinity (catalytic) site and the low-affinity site, we find that the conformation about the glycosidic bond is anti, the conformation of the ribose 3'-endo of the N type and the conformation about the ribose C4'-C5' bond either gauche-trans or trans-gauche. The values for the glycosidic bond torsion angles chi (O4'-C1'-N9-C4) for Mg2+ATP, Mg2+ADP and Mg2+AdoPP[NH]P bound to the low-affinity site of FITC-modified Ca2+ATPase are approximately equal to 270 degrees, approximately equal to 260 degrees and approximately equal to 240 degrees respectively. In the case of the nucleotides bound to the high-affinity (catalytic) site of native Ca2+ATPase, chi lies in the range 240-280 degrees.
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The kinetics of the reaction of fully reduced membrane bound cytochrome oxidase with CO following photolysis of the fully reduced cytochrome oxidase-CO complex habe been re-examined by re-analysing the data of Clore and Chance (1978) Biochem. J. 175, 709-725) at six temperatures in the 178-203 K range simultaneously at only a single wavelength pair, 444-463 nm. The choice of the 444-463 nm wavelength pair was based on the fact that the absorbance change produced at 444-463 nm on photolysis of the CO complex is sufficiently large and the separation between monitoring and reference wavelengths sufficiently small to render the effects of any possible time dependent scattering changes insignificant. On the basis of our analysis only a two step mechanism (Model 1 of Clore and Chance (1978) Biochem. J. 175, 709-725) satisfies the triple requirement of a S.D. within the standard error of the data, a random distribution of residuals and good determination of the optimized parameters. The single step mechanism of De Fonseka and Chance (1978) Biochem. J. 175, 1137-1138) fails to satisfy all three requirements. The pure difference spectra of species Ic minus E, E minus IIc and Ic minus IIc are calculated from the computed kinetics of the individual species and repetitive slow wavelength scanning difference spectra (reaction sample minus the CO complex) taken during the course of the reaction of fully reduced cytochrome oxidase with CO at 176 K.
The reaction of mixed valence state cytochrome oxidase (Cu(A) (2+)a(3+) . Cu(B) (+)a(3) (2+)) with O(2) at 173 K has been investigated in purified potato mitochondria by low temperature flash photolysis and rad wavelength scanning optical spectrometry in the visible region. The kinetics of the reaction have been analyzed simultaneously at six wavelength pairs (586-630, 590-630, 594-630, 604-630, 607-630, and 610-630 nanometers) by nonlinear optimization techniques, and found to proceed by a two-species sequential mechanism. The "pure" difference spectra of the two species, I(M) and II(M), relative to unliganded mixed valence state cytochrome oxidase have been obtained. The difference spectrum of species I(M) is characterized by a peak at 591 nanometers, with a shoulder at 584 nanometers and a trough at 602 nanometers, and that of species II(M) by an alpha band split into a prominent peak at 607 nanometers and a small side peak at 594 nanometers. Evidence is presented to suggest that these two bands arise from O(2) (-) --> Cu(B) (2+) and O(2) (-) --> a(3) (2+) charge transfer transitions which would imply that O(2) (-) forms a bridging ligand between Cu(B) and the iron atom of cytochrome a(3) in species II(M). The kinetics of the reaction and the spectral characteristics of species I(M) and II(M) obtained with the potato mitochondrial system are compared and contrasted with data in the literature on the beef heart mitochondrial system.
The 'pure' difference spectra of the three species, IM, IIM and IIIM, formed in the low-temperature reaction of membrane-bound mixed-valence-state cytochrome oxidase with O2 relative to unliganded membrane-bound mixed-valence-state cytochrome oxidase were characterized by optical spectroscopy in the visible region. The difference spectrum of species IM was characterized by a peak at 590 nm and a trough at 608 nm, that of species IIM by a peak at 606 nm, and that of species IIIM by a peak at 610 nm. A comparison with the difference spectra of species IIM and IIIM obtained with soluble cytochrome oxidase [Clore, Andréasson, Karlsson, Aasa & Malmström (1980) Biochem. J. 185, 155-167] revealed small but significant differences in the peak positions and bandwidths of the 605-610 nm absorption band.
The kinetics and thermodynamics of the reaction of mixed valence state membrane-bound cytochrome oxidase with CO over the 178-203 K range has been studied by multichannel optical spectroscopy at three wavelength pairs (444-463 nm in the Soret region, and 590-630 and 608-630 nm in the alpha region) and analysed by non-linear optimization techniques. As in the case of the fully reduced membrane-bound cytochrome oxidase-CO reaction (Clore, G.M. and Chance, E.M. (1978) Biochem J. 175, 709-725), the normalized progress curves at the three wavelength pairs are significantly different indicating, on the basis of Beer's law, the presence of a minimum of three optically distinct species. The only model that satisfies the triple statistical requirement of a standard deviation within the standard error of the data, a random distribution of residuals and good determination of the optimized parameters, is a two species sequential mechanism: flash photolysis of the mixed valence state cytochrome oxidase-CO complex (species IIMC) yields unliganded mixed valence state cytochrome oxidase (species EM) and free CO which then recombine to form species IMC; species IMC is then converted into species IIMC. All the thermodynamic parameters describing the model are calculated and compared to those obtained for the fully reduced membrane-bound cytochrome oxidase-CO reaction (Clore and Chance (1978) Biochem. J. 175, 709-725). Although there are some qualitative similarities in the kinetics and thermodynamics of the reactions of mixed valence state (alpha 23+Cu+B.ALPHA 3+Cu2+A) and fully reduced (a3 2+Cu B + . a2+Cu A+) cytochrome oxidase with CO, there are large and significant quantitative differences in zero-point activation energies and frequency factors; over the temperature range studied, the mixed valence state cytochrome oxidase-CO reaction is found to proceed at a significantly slower rate than the fully reduced cytochrome oxidase-CO reaction. These differences indicate that changing the valence states of cytochrome a and CuA has a significant effect on the CO binding properties of cytochrome a 3 and possibly CuB.
The reaction of fully reduced soluble bovine heart cytochrome oxidase with O2 at 173K was investigated by low-temperature optical and e.p.r. spectroscopy, and the kinetics of the reaction were analysed by non-linear optimization techniques. The only e.p.r. signals seen during the course of the reaction are those attributable to low-spin cytochrome a3+ and CuA2+. Quantitative analysis of e.p.r. signals shows that, at the end point of the reaction at 173K, nearly 100% of CuA is in the cupric state but only about 40% of cytochrome a is in the ferric low-spin state. The optical spectra recorded at this stage of the reaction show incomplete oxidation of haem and the absence of a 655 nm absorption band. The only reaction scheme that accounts for both the e.p.r. and optical data is a four-intermediate mechanism involving a branching pathway. The reaction is initiated when fully reduced cytochrome oxidase reacts with O2 to form intermediate I. This is then converted into either intermediate IIA or intermediate IIB. Of these, intermediate IIB is a stable end product at 173 K, but intermediate IIA is converted into intermediate III, which is the stable state at 173 K in this branch of the mechanism. The kinetic analysis of the e.p.r. data allows the unambiguous assignments of the valence states of cytochrome a and CuA in the intermediates. Intermediate I contains cytochrome a2+ and CuA+, intermediate IIA contains low-spin cytochroma a3+ and CuA+, intermediate IIB contains cytochrome a2+ and CuA2+, and intermediate III contains low-spin cytochrome a3+ and CuA2+. The electronic state of the O2-binding CuBa3 couple during the reoxidation of cytochrome oxidase is discussed in terms of an integrated structure containing CuB, cytochrome a3 and O2.
The reaction of soluble mixed-valence-state (a3+CuA 2+.CuB + A32+) cytochrome oxidase with O2 at low temperature was studied by optical and e.p.r. spectroscopy. The existence of three intermediates [Clore & Chance (1978) Biochem. J. 173, 799-8101] was confirmed. From the e.p.r data it is clear that cytochrome a and CuA remain in the low-spin ferric and cupric states respectively throughout the reaction. No e.p.r. signals attributable to cytochrome a3 or CuB were seen in the intermediates. The difference spectra (intermediates minus unliganded mixed-valence-state cytochrome oxidase) and absolute spectra of the three intermediates were obtained. The chemcal nature of the three intermediates is discussed in terms of their spectroscopic properties. A catalytic cycle for cytochrome oxidase is proposed.
The existence of a temperature-induced absorption band centred in the region of 666 nm is demonstrated for both membrane-bound and soluble cytochrome oxidase in the frozen state. The 666 nm band is generated solely by an increase in temperature of both fully reduced and mixed valence state cytochrome oxidase in the presence of CO or O2 within the 'pocket' containing the active site; it is not formed in the absence of both CO and O2 from the sample. The formation of the 666 nm band is entirely reversible when the temperature is decreased again and its formation is not dependent on the presence of liganded CO at the sixth coordination site of haem a3 in the low temperature range (below --120 degrees C) prior to photolysis. The shape and intensity of the 666 nm band are not affected by the extent of CO recombination following flash and photolysis and temperature increase and are not affected by changes in the valence states of the four metal centres when the O2 reaction is in progress.
The kinetics of the reaction of fully reduced membrane-bound cytochrome oxidase with O2 obtained in the Soret, alpha and near-i.r. regions were analysed, and the contributions of the three intermediates of the reaction [Clore & Chance (1978) Biochem. J. 173, 799--810] to seven wavelength pairs (430--463, 444--463, 590--630, 608--630, 740--940, 790--940 and 830--940 nm) were determined. The nature of the intermediates is discussed on the basis of the data in the present paper together with data in the literature from optical wavelength scanning, e.p.r., i.r. and magnetic-susceptibility studies.
1. The results of non-linear optimization studies on the mechanism of reaction of fully reduced cytochrome oxidase with O2 at 176K are presented. The analysis is carried out on data obtained by means of dual-wavelength multi-channel spectroscopy at three wavelength pairs (604-630, 608-630 and 830-940 nm) and at three O2 concentrations (60, 200 and 1180 micron). The only model that satisfies the triple requirement of a standard deviation within the standard error of the experimental data, good determination of the optimized parameters and a random distribution of residuals is a three-species sequential mechanism. 2. On the basis of the optimized values of the relative absorption coefficients of the intermediates at each wavelength obtained from the present paper together with data from low-temperature trapping, e.p.r. and magnetic-susceptibility studies, the possible valence states of the metal centres in each of the intermediates are discussed.
1. The results of non-linear optimization studies on the mechanism of reaction of ferricyanide-pretreated mixed-valence-state cytochrome oxidase with O2 at 173 K are presented. The analysis is carried out on data obtained by means of dual-wavelength multi-channel spectroscopy at four wavelength pairs (444-463 nm, 604-630 nm, 608-630 nm and 830-940 nm) and at two O2 concentrations (360 micron and 520 micron). The only model that satisfies the triple requirement of a standard deviation within the standard error of the experimental data, a random distribution of residuals and good determination of the optimized parameters, is a three-intermediate sequential mechanism. 2. On the basis of the optimized values of the relative absorption coefficients of the intermediates at each wavelength obtained from the present paper together with data from optical wavelength scanning and e.p.r. spectroscopy obtained by low-temperature trapping studies, the possible valence states of the metal centres in each of the intermediates are discussed.
A search procedure is described for making stereospecific assignments at prochiral centers in proteins on the basis of nuclear Overhauser enhancement and coupling constant data derived from nmr experiments. A data base comprising torsion angles, associated 1H-1H coupling constants and interproton distances is searched by a computer algorithm for sets of values that match the experimental data within specified error limits. Two different data bases are used. The first is a crystallographic data base derived from 34 well-refined crystal structures; the second is a systematic data base derived from conformations of a short peptide fragment with idealized geometry by systematically varying the phi, psi, and chi 1 torsion angles. Both approaches are tested for beta-methylene groups with model data obtained from 20 crystal structures. The results for the two methods are similar though not identical, so that a combination of the two methods appears to be useful. With an appropriate choice of error estimates, around 80% of the beta-methylene groups could be assigned in the test calculations. In addition, results with experimental nmr data indicate that a similar percentage of stereospecific assignments can be made in practical situations.
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The solution structure of the 64 residue structured domain (residues 20-83) of barley serine proteinase inhibitor 2 (BSPI-2) is determined on the basis of 403 interproton distance, 34 phi backbone torsion angle and 26 hydrogen bonding restraints derived from n.m.r. measurements. A total of 11 converged structures were computed using a metric matrix distance geometry algorithm and refined by restrained molecular dynamics. The average rms difference between the final 11 structures and the mean structure obtained by averaging their coordinates is 1.4 +/- 0.2 A for the backbone atoms and 2.1 +/- 0.1 A for all atoms. The overall structure, which is almost identical to that found by X-ray crystallography, is disc shaped and consists of a central four component mixed parallel and antiparallel beta-sheet flanked by a 13 residue alpha-helix on one side and the reactive site loop on the other.