5 K extended X-ray absorption fine structure and 40 K 10-s resolved extended X-ray absorption fine structure studies of photolyzed carboxymyoglobin

A previous extended X-ray absorption fine structure (EXAFS) study of photolyzed carboxymyoglobin (MbCO) [Chance, B., Fischetti, R., & Powers, L. (1983) Biochemistry 22, 3820-3829; Powers, L., Sessler, J. L., Woolery, G. L., & Chance, B. (1984) Biochemistry 23, 5519-5523] has provoked much discussion on the heme structure of the photoproduct (MbCO). The EXAFS interpretation that the Fe-CO distance increases by no more than 0.05 A following photodissociation has been regarded as inconsistent with optical, infrared, and magnetic susceptibility studies [Fiamingo, F. G., & Alben, J. O. (1985) Biochemistry 24, 7964-7970; Sassaroli, M., & Rousseau, D. L. (1986) J. Biol. Chem. 261, 16292-16294]. The present experiment was performed with well-characterized dry film samples in which MbCO molecules were embedded in a poly(vinyl alcohol) matrix [Teng, T. Y., & Huang, H. W. (1986) Biochim. Biophys. Acta 874, 13-18]. The sample had a high protein concentration (12 mM) to yield adequate EXAFS signals but was very thin (40 micron) so that complete photolysis could be easily achieved by a single flash from a xenon lamp. Although the electronic state of MbCO resembles that of deoxymyoglobin (deoxy-Mb), direct comparison of EXAFS spectra indicates that structurally MbCO is much closer to MbCO than to deoxy-Mb.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome c peroxidase compound ES is identical with horseradish peroxide compound I in iron-ligand distances

X-ray absorption studies of compound ES of cytochrome c peroxidase show a short iron-oxygen distance of 1.67 +/- 0.04 A, an iron-histamine distance of 1.91 +/- 0.03 A, and an iron-pyrrole nitrogen average distance of 2.02 +/- 0.02 A. This is identical within the error with the reported structure of horseradish peroxidase compound I [Chance, B., Powers, L., Ching, Y., Poulos, T., Yamazaki, I., & Paul, K. G. (1984) Arch. Biochem. Biophys. 235, 596-611]. Comparisons of the structures of myoglobin peroxide [Chance, M., Powers, L., Kumar, C., & Chance, B. (1986) Biochemistry (preceding paper in this issue)], compound ES, and the intermediates of horseradish peroxidase reveal the possible mechanisms for the stabilization of the free radical species generated during catalysis. The proximal histidine regulates the structure and function of the pyrrole nitrogens and the heme, allowing for the formation and maintenance of the characteristic intermediates.     

Structural aspects of the copper sites in cytochrome c oxidase. An X-ray absorption spectroscopic investigation of the resting-state enzyme

Copper K-edge X-ray absorption spectroscopy (XAS) has been used to investigate the structural details of the coordination environment of the copper sites in eight resting-state samples of beef heart cytochrome c oxidase prepared by different methods. The unusual position and structure of the resting-state copper edge spectrum can be adequately explained by the presence of sulfur-containing ligands, with a significant amount of S----Cu(II) charge transfer (i.e., a covalent site). Quantitative curve-fitting analysis of the copper extended X-ray absorption fine structure (EXAFS) data indicates similar average first coordination spheres for all resting-state samples, regardless of preparation method. The average coordination sphere (per 2 coppers) mainly consists of 6 +/- 1 nitrogens or oxygens at an average Cu-(N,O) distance of 1.99 +/- 0.03 A and 2 +/- 1 sulfurs at an average Cu-S distance of 2.28 +/- 0.02 A. Quantitative curve-fitting analysis of the outer shell of the copper EXAFS indicates the presence of a Cu...Fe interaction at a distance of 3.00 +/- 0.03 A. Proposed structures of the two copper sites based on these and other spectroscopic results are presented, and differences between our results and those of other published copper XAS studies [Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465-498] are discussed.     

"Peroxidatic" form of cytochrome oxidase as studied by X-ray absorption spectroscopy

X-ray absorption spectroscopy shows pulsed oxidase to be similar to resting oxidase but to lack the sulfur bridge between iron and copper of active sites (Powers, L., Y. Ching, B. Chance, and B. Muhoberac, 1982, Biophys. J., 37[2, Pt. 2]: 403a. [Abstr.] ) The first shell ligands and bond lengths of the pulsed oxidase active site heme most clearly fit the ferric peroxidases from horseradish and yeast, and the pulsed oxidase cyanide compound resembles the low spin hemoprotein cyanide compounds. The structural results are consistent with an aquo or a peroxo form for pulsed oxidase as is also observed by optical studies. These structural and chemical data are consistent with a role for the pulsed forms in a cyclic peroxidatic side reaction in which the pulsed and pulsed peroxide compounds act as peroxide scavengers. The peroxidatic role of cytochrome oxidase in the nonsulfur bridged form suggests the renaming of the "oxygenated" or "pulsed" forms on a functional basis as "peroxidatic" forms of cytochrome oxidase.     

A spectral study of cobalt(II)-substituted Bacillus cereus phospholipase C

The coordination sphere of both the structural and catalytic zinc ions of Bacillus cereus phospholipase C has been probed by substitution of cobalt(II) for zinc and investigation of the resultant derivatives by a variety of spectroscopic techniques. The electronic absorption, circular dichroic, magnetic circular dichroic, and electron paramagnetic resonance spectra were found to be strikingly similar when cobalt(II) was substituted into either site and are consistent with a distorted octahedral environment for the metal ion in both sites. Octahedral coordination appears comparatively rare in zinc metalloenzymes but has been suggested for glyoxalase I [Sellin, S., Eriksson, L. E. G., Aronsson, A.-C., & Mannervik, B. (1983) J. Biol. Chem. 258, 2091-2093; Garcia-Iniguez, L., Powers, L., Chance, B., Sellin, S., Mannervik, B., & Mildvan, A. S. (1984) Biochemistry 23, 685-689], transcarboxylase [Fung, C.-H., Mildvan, A. S., & Leigh, J. S. (1974) Biochemistry 13, 1160-1169], and the regulatory binding site of Aeromonas aminopeptidase [Prescott, J. M., Wagner, F. W., Holmquist, B., & Vallee, B. L. (1985) Biochemistry 24, 5350-5356]. Phospholipase C is so far unique in having two such sites.     

Kinetic, structural, and spectroscopic identification of geminate states of myoglobin: a ligand binding site on the reaction pathway

Elementary steps or geminate states in the reaction of gaseous ligands with transport proteins delineate the trajectory of the ligand and its rebinding to the heme. By use of kinetic studies of the 765-nm optical "conformation" band, three geminate states were identified for temperatures less than approximately 100 K. MbCO, which is accumulated by photolysis between 1.2 and approximately 10 K, was characterized by our previous optical and X-ray absorption studies [Chance, B., Fischetti, R., & Powers, L. (1983) Biochemistry 22, 3820-3829]. Between 10 and approximately 100 K, geminate states that are also identified that have recombination rates of approximately 10(3) s-1 and approximately 10(-5) s-1 (40 K). Thus, it is possible to maintain a steady-state nearly homogeneous population of the slowest recombining geminate state, Mb, by regulated continuous illumination (optical pumping). Both X-ray absorption and resonance Raman studies under similar conditions of optical pumping show that the heme structure around the iron in Mb is similar to that of MbCO. In both geminate states, the iron-proximal histidine distance remains unchanged (+/- 0.02 A) from that of MbCO while the iron to pyrrole nitrogen average distance has not fully relaxed to that of the deoxy state. In MbCO the CO remains close to iron but not bound, and the Fe...CO angle, which is bent in MbCO (127 +/- 4 degrees C), is decreased by approximately 15 degrees [Powers, L., Sessler, J. L., Woolery, G. L., & Chance, B. (1984) Biochemistry 23, 5519-5523]. The CO molecule in Mb, however, has moved approximately 0.7 A further from iron. Computer graphics modeling of the crystal structure of MbCO places the CO in a crevice in the heme pocket that is just large enough for the CO molecule end-on. Above approximately 100 K resonance Raman studies show that this structure relaxes to the deoxy state.     

X-ray absorption studies of myoglobin peroxide reveal functional differences between globins and heme enzymes

X-ray absorption studies of myoglobin peroxide show that although it is not identical with compound I or II of horseradish peroxidase [Chance, B., Powers, L., Ching, Y., Poulos, T., Yamazaki, I., & Paul, K. G. (1984) Arch. Biochem. Biophys. 235, 596-611], it has some structural features in common with both. As seen in compound I, the Fe-O distance is short, but the iron-pyrrole nitrogen distance is contracted with a longer iron-histidine distance like compound II. The iron has a higher oxidation state than Fe3+, suggesting an oxyferryl ion type species. Comparison of the structures of various peroxidase and myoglobin compounds points out systematic differences that may explain the catalytic activity of the pi cation radical as well as some of the differences between globins and heme enzymes.     

Structure and reactivity of multiple forms of cytochrome oxidase as evaluated by X-ray absorption spectroscopy and kinetics of cyanide binding

The extended X-ray absorption fine structure (EXAFS) data show differences between the active site structures of different cytochrome oxidase preparations. In the resting (as isolated) state of the Yonetani preparation, the bridging atom between Fe3+a3 and Cu2+a3 is present [Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465], whereas in another preparation (e.g., Hartzell-Beinert), this atom seems to be bound only to Fe3+a3 in a significant fraction of the molecules. Both preparations bind cyanide in a multiphasic fashion, suggesting that the resting cytochrome oxidase is not homogeneous but rather is a mixture of several forms. The proportion of these forms as detected by cyanide binding kinetics differs for different preparations. However, upon reduction and reoxidation (conversion to the "oxygenated" form) the cyanide binding kinetics become monophasic and all preparations of the oxygenated form bind cyanide at the same rate. Thus, a combination of structural and kinetic approaches seems necessary for evaluation of the nature of the active site of cytochrome oxidase in its various forms.     

CO bond angle changes in photolysis of carboxymyoglobin

Previous studies [Chance, B., Fischetti, B., & Powers, L. (1983) Biochemistry 22, 3820-3829] of the local structure changes around the iron in carboxymyoglobin on photolysis at 4 K revealed that the iron-carbon distance increased approximately 0.05 A but was accompanied by a lengthening of the iron-pyrrole nitrogen bonds of the heme (approximately 0.03 A) that was not as large as that found in the deoxy form. Further analysis of these data together with comparison to model compounds indicates that the Fe-C-O bond angle in carboxymyoglobin is bent (127 +/- 4 degrees), having a structure identical, within the error, with the "pocket" porphyrin model compound FePocPiv(1-MeIm)(CO) [Collman, J. P., Brauman, J. I., Collins, T. J., Iverson, B. L., Lang, G., Pettman, R., Sessler, J. L., & Walters, M. A. (1983) J. Am. Chem Soc. 105, 3038-3052]. On photolysis, this angle decreases by 5-10 degrees. In addition, correlation is observed between the increase in the length of the Fe-C bond and the decrease of the Fe-C-O angle. These results suggest that the rate-limiting step in recombination is the thermal motion of CO in the pocket to achieve an appropriate bonding angle with respect to the iron. These changes constitute the first molecular picture of the photolysis process, as well as the structure of the geminate state, and are important in clarifying nuclear tunneling parameters.     

Structures of photolyzed carboxymyoglobin

The structures of photoactivated carboxymyoglobin (Mb*CO) at temperatures to 10 K have been investigated by Fourier transform infrared (FT-IR) spectroscopy, visible spectroscopy, and near-infrared spectroscopy. Two energy states for *CO are observed by FT-IR, which are altered in frequency by 94% and 88% of the difference from the ground-state heme CO toward free CO gas [Alben, J. O., Beece, D., Bowne, S. F., Doster, W., Eisenstein, L. Frauenfelder, H., Good, D., McDonald, J. D., Marden, M. C., Moh, P. P., Reinisch, L., Reynolds, A. H., Shyamsundar, E., & Yue, K. T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3744-3748]. Ground-state MbCO shows no absorption in the near-infrared from 700 to 1200 nm. Conversely, Mb*CO shows an absorption near 766 nm, similar to that of ferrous myoglobin (deoxy-Mb) at 758 nm. These data are compared with M?ssbauer isomer shifts and quadrupole splitting [Spartalian, K., Lang, G., & Yonetani, T. (1976) Biochim. Biophys. Acta 428, 281-290] and magnetic susceptibility measurements [Roder, H., Berendzen, J., Bowne, S. F., Frauenfelder, H., Sauke, T. B., Shyamsunder, E., & Weissman, M. B. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2359-2363], which clearly indicate that the iron in both Mb*CO and deoxy-Mb is in the high-spin Fe(II) state, as does the heme transition in the Soret [Iizuka, T., Yamamoto, H., Kotani, M., & Yonetani, T. (1974) Biochim. Biophys. Acta 371, 126-139]. Thus the electronic structure of iron in Mb*CO is nearly identical with that of deoxy-Mb, and *CO is only slightly perturbed from the free gas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calculation of free-Mg2+ concentration in adenosine 5'-triphosphate containing solutions in vitro and in vivo

We have attempted to resolve the differences between the levels of free Mg2+ in muscle calculated by Wu et al. [Wu, S. T., Pieper, G. M., Salhany, J. M., & Eliot, R. S. (1981) Biochemistry 20, 7399-7403] (2.5 mM in guinea pig heart) and by Gupta and Moore [Gupta, R. K., & Moore, R. D. (1980) J. Biol. Chem. 255, 3987-3993] (0.6 mM in frog skeletal muscle) on the basis of substantially identical measurements by 31P NMR of the phosphate peaks in the spectrum of MgATP2-. The differences depend on the methods of calculation, including which reactions in which multiple equilibria are being considered. Biochemists and physical chemists customarily use different working definitions of the stability constant for MgATP2- in particular. Wu et al. used in their calculations, without reconciliation, methods involving three different operational definitions of the chelation equilibria involved. An algorithm for calculating Mg2+ and total ATP, which can be carried out with a hand calculator, is described here. With it, we calculated Mg2+ levels that agree with those determined by Gupta et al. [Gupta, R. K., Benkovic, J. L., & Rose, Z. B. (1978) J. Biol. Chem. 253, 6165-6171] with their in vitro systems. We therefore agree with the finding of Gupta and Moore that the Mg2+ level in skeletal and cardiac muscle is 0.6 mM.     

X-ray absorption spectroscopic study of the active copper sites in dopamine beta-hydroxylase

X-ray absorption spectroscopy has been used to investigate the local environment of the copper sites in bovine dopamine beta-hydroylase, the enzyme that catalyzes the conversion of dopamine to norepinephrine in the adrenal medulla and noradrenergic nerve cells. The marked similarity of the x-ray absorption edge features of the oxidized and ascorbate-reduced forms of the enzyme with those of the corresponding Cu(imidazole)4 complexes suggests that the ligation in both cases is very similar. Furthermore, this similarity is found for the extended x-ray absorption fine structure data, and analysis shows only nitrogen (or oxygen) ligation for both enzyme forms. Thus, four nitrogen atoms provide the best fit to the data at an average distance of 1.97 +/- 0.02 A for the oxidized enzyme and four nitrogen atoms at 2.05 +/- 0.02 A for the ascorbate-reduced form. The present data analysis also indicates that there is little change in the average copper ligand environment upon reduction of the enzyme-bound copper from Cu(II) to the Cu(I). The data for the oxidized form of the enzyme are in agreement with previous spin-echo EPR experiments that show three to four imidazole nitrogen ligands for each copper (McCracken, J., Desai, P. R., Papadopoulos, N. J., Villafranca, J. J., and Peisach, J. (1988) Biochemistry 27, 4133-4137). In addition, the data do not indicate the presence of any heavy atom (sulfur or chlorine) ligation to the ascorbate-reduced form of the enzyme as reported by Scott et al. (Scott, R. A., Sullivan, R. J., DeWolf, W. E., Jr., Dolle, R. E., and Kruse, L. I. (1988) Biochemistry 27, 5411-5417).     

Role of histone tails in the conformation and interactions of nucleosome core particles

The goal of this work was to test the role of the histone tails in the emergence of attractive interactions between nucleosomes above a critical salt concentration that corresponds to the complete tail extension outside the nucleosome [Mangenot, S., et al (2002) Biophys. J. 82, 345-356; Mangenot, S., et al (2002) Eur. Phys. J. E 7, 221-231]. Small angle X-ray scattering experiments were performed in parallel with intact and trypsin tail-deleted nucleosomes with 146 +/- 3 bp DNA. We varied the monovalent salt concentration from 10 to 300 monovalent salt concentration and followed the evolution of (i) the second virial coefficient that characterizes the interactions between particles and (ii) the conformation of the particle. The attractive interactions do not emerge in the absence of the tails, which validates the proposed hypothesis.     

Morphology and dynamics of chromosome territories in living cells.

Chromosome territories formed by fluorescence-labeled sub-chromosomal foci were analyzed in time-lapse series of 3D confocal data sets of living HeLa and human neuroblastoma cells. The quantitative analysis of the chromosome territory morphology confirmed previous results obtained by visual observation [Zink et al., Hum. Genet. 102 (1998) 241-251] that chromosome territories persisted as stable entities over an observation time >4 h. The changes in morphology with time of single chromosome territories were found to be less pronounced than differences in morphology of different chromosome territories in fixed cells. The analysis of the individual motion of chromosome territories recently showed 'Brownian' diffusion-like motion at very slow rates [Bornfleth et al., Biophys. J. 77 (1999) 2871-2886]. Here, we show that the mutual motion of different chromosome territories was independent and also 'Brownian' diffusion-like.     

Morphology and dynamics of chromosome territories in living cells

Chromosome territories formed by fluorescence-labeled sub-chromosomal foci were analyzed in time-lapse series of 3D confocal data sets of living HeLa and human neuroblastoma cells. The quantitative analysis of the chromosome territory morphology confirmed previous results obtained by visual observation [Zink et al., Hum. Genet. 102 (1998) 241–251] that chromosome territories persisted as stable entities over an observation time >4 h. The changes in morphology with time of single chromosome territories were found to be less pronounced than differences in morphology of different chromosome territories in fixed cells. The analysis of the individual motion of chromosome territories recently showed ‘Brownian’ diffusion-like motion at very slow rates [Bornfleth et al., Biophys. J. 77 (1999) 2871–2886]. Here, we show that the mutual motion of different chromosome territories was independent and also ‘Brownian’ diffusion-like.     

Glutamic acid-149 is important for enzymatic activity of yeast inorganic pyrophosphatase

Modification of Saccharomyces cerevisiae inorganic pyrophosphatase (PPase) with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide is known to lead to a loss of enzymatic activity, the rate of which is decreased in the presence of ligands binding to the active site [Cooperman, B. S., & Chiu, N. Y. (1973) Biochemistry 12, 1676-1682; Heitman, P., & Uhlig, H. J. (1974) Acta Biol. Med. Ger. 32, 565-594]. In this work we show that, when such inactivation is carried out in the presence of [14C]glycine ethyl ester (GEE), GEE is covalently incorporated into PPase, incorporation into the most highly labeled tryptic peptide is site-specific, as evidenced by the reduction of such incorporation in the presence of the active site ligands Zn2+ and Pi, the extent of formation of this specifically labeled peptide correlates with the fractional loss of PPase activity, and the specifically labeled peptide corresponds to residues 145-153 and the position of incorporation within this peptide is Glu-149. The significance of our findings for the location of the active site and for the catalytic mechanism of PPase is briefly considered in the light of the 3-A X-ray crystallographic structure of Arutyunyun and his colleagues [Arutyunyun, E. G., et al. (1981) Dokl. Akad. Nauk SSSR 258, 1481-1485; Kuranova, I. P., et al. (1983) Bioorg. Khim. 9, 1611-1919; Terzyan, S. S., et al. (1984) Bioorg. Khim. 10, 1469-1482].     

The Stanford Microarray Database

The Stanford Microarray Database (SMD) stores raw and normalized data from microarray experiments, and provides web interfaces for researchers to retrieve, analyze and visualize their data. The two immediate goals for SMD are to serve as a storage site for microarray data from ongoing research at Stanford University, and to facilitate the public dissemination of that data once published, or released by the researcher. Of paramount importance is the connection of microarray data with the biological data that pertains to the DNA deposited on the microarray (genes, clones etc.). SMD makes use of many public resources to connect expression information to the relevant biology, including SGD [Ball,C.A., Dolinski,K., Dwight,S.S., Harris,M.A., Issel-Tarver,L., Kasarskis,A., Scafe,C.R., Sherlock,G., Binkley,G., Jin,H. et al. (2000) Nucleic Acids Res., 28, 77-80], YPD and WormPD [Costanzo,M.C., Hogan,J.D., Cusick,M.E., Davis,B.P., Fancher,A.M., Hodges,P.E., Kondu,P., Lengieza,C., Lew-Smith,J.E., Lingner,C. et al. (2000) Nucleic Acids Res., 28, 73-76], Unigene [Wheeler,D.L., Chappey,C., Lash,A.E., Leipe,D.D., Madden,T.L., Schuler,G.D., Tatusova,T.A. and Rapp,B.A. (2000) Nucleic Acids Res., 28, 10-14], dbEST [Boguski,M.S., Lowe,T.M. and Tolstoshev,C.M. (1993) Nature Genet., 4, 332-333] and SWISS-PROT [Bairoch,A. and Apweiler,R. (2000) Nucleic Acids Res., 28, 45-48] and can be accessed at http://genome-www.stanford.edu/microarray.     

Autophagy and p70S6 Kinase

A paper by Scott et al.,1 suggested that p70S6 kinase (p70S6k) is a positive regulatory factor for autophagy. This finding is in contrast to previous data suggesting a negative role for this factor. The Scott et al. article was highlighted in Nature News & Views,2 which elicited a commentary by A.J. Meijer and P. Codogno. These authors present an alternate model for the role of p70S6k in autophagic induction, although still as a positive factor. Following the initial commentary is a response by T.P. Neufeld and R.C. Scott.     

Phospholipid fatty acyl chain asymmetry in the membrane bilayer of isolated skeletal muscle sarcoplasmic reticulum

We previously showed [Herbette, L. G., Blasie, J. K., DeFoor, P., Fleischer, S., Bick, R. J., Van Winkle, W. B., Tate, C. A., & Entman, M. L. (1984) Arch. Biochem. Biophys. 234, 235-242; Herbette, L. G., DeFoor, P., Fleischer, S., Pascolini, D., Scarpa, A., & Blasie, J. K. (1985) Biochim. Biophys. Acta 817, 103-122] that the phospholipid head-group distribution in the membrane bilayer of isolated sarcoplasmic reticulum is asymmetric. From these studies, both the total number of phospholipid head groups and the total lipid, as well as the head-group species for these lipids, were found to be different for each monolayer of the membrane bilayer. In this paper, we demonstrate for the first time that there is significant asymmetry in the distribution of unsaturated fatty acids between the two monolayers; i.e., the outer monolayer of the sarcoplasmic reticulum contained more unsaturated and polyunsaturated chains when compared to the inner monolayer. X-ray diffraction measurements demonstrated that the time-averaged fatty acyl chain extension for the outer monolayer was approximately 20% less than for the inner monolayer. This is consistent with the concept that the greater degree of unsaturation in the outer monolayer may provide for a decreased average fatty acyl chain extension for that layer. This architecture for the bilayer may be related to both the "resting" state mass distribution of the calcium pump protein within the membrane bilayer and possible "conformational" states of the calcium pump protein during calcium transport by the sarcoplasmic reticulum.