Functional consequences of single:double ring transitions in chaperonins: life in the cold

The cpn60 and cpn10 genes from psychrophilic bacterium, Oleispira antarctica RB8, showed a positive effect in Escherichia coli growth at low temperature, shifting its theoretical minimal growth temperature from +7.5 degrees C to -13.7 degrees C [Ferrer, M., Chernikova, T.N., Yakimov, M., Golyshin, P.N., and Timmis, K.N. (2003) Nature Biotechnol 21: 1266-1267]. To provide experimental support for this finding, Cpn60 and 10 were overproduced in E. coli and purified to apparent homogeneity. Recombinant O.Cpn60 was identical to the native protein based on tetradecameric structure, and it dissociates during native PAGE. Gel filtration and native PAGE revealed that, in vivo and in vitro, (O.Cpn60)(7) was the active oligomer at 4-10 degrees C, whereas at > 10 degrees C, this complex was converted to (O.Cpn60)(14). The dissociation reduces the ATP consumption (energy-saving mechanism) and increases the refolding capacity at low temperatures. In order for this transition to occur, we demonstrated that K468 and S471 may play a key role in conforming the more advantageous oligomeric state in O.Cpn60. We have proved this hypothesis by showing that single and double mutations in K468 and S471 for T and G, as in E.GroEL, produced a more stable double-ring oligomer. The optimum temperature for ATPase and chaperone activity for the wild-type chaperonin was 24-28 degrees C and 4-18 degrees C, whereas that for the mutants was 45-55 degrees C and 14-36 degrees C respectively. The temperature inducing unfolding (T(M)) increased from 45 degrees C to more than 65 degrees C. In contrast, a single ring mutant, O.Cpn60(SR), with three amino acid substitutions (E461A, S463A and V464A) was as stable as the wild type but possessed refolding activity below 10 degrees C. Above 10 degrees C, this complex lost refolding capacity to the detriment of the double ring, which was not an efficient chaperone at 4 degrees C as the single ring variant. We demonstrated that expression of O.Cpn60(WT) and O.Cpn60(SR) leads to a higher growth of E. coli at 4 degrees C ( micro (max), 0.22 and 0.36 h(-1) respectively), whereas at 10-15 degrees C, only E. coli cells expressing O.Cpn60 or O.Cpn60(DR) grew better than parental cells (-cpn). These results clearly indicate that the single-to-double ring transition in Oleispira chaperonin is a wild-type mechanism for its thermal acclimation. Although previous studies have also reported single-to-double ring transitions under many circumstances, this is the first clear indication that single-ring chaperonins are necessary to support growth when the temperature falls from 37 degrees C to 4 degrees C.     

Functional differentiation in trematode hemoglobin isoforms.

The Hbs and the major electrophoretic Hb components (isoHbs) were isolated from three species of the trematodes, Explanatum explanatum (Ee), Gastrothylax crumenifer (Gc) and Paramphistomum epiclitum (Pe), that parasitise the common Indian water buffalo Bubalus bubalis. The Hbs are monomeric and resemble the so-called nonfunctional mutant hemoglobins that have Tyr at B10 or E7 positions (replacing Leu and the His residues, respectively). However, they are capable of binding with O2 and CO. O2 equilibrium studies of trematode Hb isoforms reveal extremely high O2 affinities, with half-saturation O2 tension (P50) values up to 800 times lower than those of human hemoglobins. This correlates with Tyr residues at B10 and at the distal position (E7) that decrease the O2 dissociation rate by contributing hydrogen bonds (H-bonds) to the bound O2. These substitutions also increase the O2 association rates either due to orientation of E7-Tyr towards the solvent and/or by sterically hindering the entry of water molecules into the heme pocket. The latter may account for the low rate of autoxidation of trematode Hbs. The Hbs and their isoforms from different species exhibited pronounced variation in O2 affinity, which may relate to subtle differences in the structure of the heme pocket. The O2 affinities of the composite (unfractionated) Hbs were intermediate to those of the individual Hb isoform. The P50 values of Hbs here obtained by direct O2 equilibrium measurements differed from those calculated from kinetic data already published [Kiger, L., Rashid, A. K., Griffon, N., Haque, M., Moens, L.,Gibson, Q. H., Poyart, C., & Marden, M. C. (1998). Biophys. J. 75, 990-998.] Intermediate state(s) due to slow reorientation of E7-Tyr may account for this difference. Some Hb isoforms showed slight (either normal or reverse) Bohr effects. The hyperbolic O2 equilibrium curve, Hill coefficient (n) values near unity accord with a monomeric nature of trematode Hbs. In marked contrast to vertebrate Hbs, CO does not seem to compete effectively with O2 in trematode Hbs, as evident from partition coefficient values (M) below 1.     

X-ray absorption spectroscopic studies of the high-spin iron(II) active site of isopenicillin N synthase: evidence for Fe-S interaction in the enzyme-substrate complex.

Isopenicillin N synthase from Cephalosporium acremonium (IPNS; M(r) 38.4K) is an Fe(2+)-requiring enzyme which catalyzes the oxidative conversion of (L-alpha-amino-delta-adipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N, with concomitant reduction of O2 to 2H2O. Chemical and spectroscopic data have suggested that catalysis proceeds via an enzyme complex of ACV bound to the iron through its cysteinyl thiolate [Baldwin, J. E., & Abraham, E. P. (1988) Nat. Prod. Rep. 5, 129-145; Chen, V. J., Orville, A. M., Harpel, M. R., Frolik, C. A., Surerus, K. K., Münck, E., & Lipscomb, J. D. (1989) J. Biol. Chem. 264, 21677-21681; Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C. A., & Chen, V. J. (1991) Biochemistry 30, 11653-11659]. Here we have employed the technique of Fe K-edge extended X-ray absorption fine structure (EXAFS) to characterize the iron site and to seek direct evidence for or against the formation of an Fe-S interaction upon ACV binding. Our data collected in the absence of substrate and O2 are consistent with the iron center of IPNS being coordinated by only (N,O)-containing ligands in an approximately octahedral arrangement and with an average Fe-(N,O) distance of 2.15 +/- 0.02 A. Upon anaerobic binding of ACV, the iron coordination environment changes considerably, and the associated Fe EXAFS cannot be adequately simulated without incorporating an Fe-S interaction at 2.34 +/- 0.02 A along with four or five Fe-(N,O) interactions at 2.15 +/- 0.02 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP induces the release of a neutral serine proteinase and enhances the production of superoxide anion in membranes from phorbol ester-activated neutrophils

Plasma membranes isolated from human neutrophils after brief exposure to phorbol 12-myristate 13-acetate contain a large portion (30-40%) of the total cellular protein kinase C (Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., and Horecker, B. L. (1986) Biochem. Biophys. Res. Commun. 136, 228-234) and also retain almost 90% of their content of neutral serine proteinase (Pontremoli, S., Melloni, E., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., Damiani, G., and Horecker, B. L. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1685-1689). When ATP is added to the isolated membranes, a substantial amount (approximately 25%) of the intrinsic proteinase is released into the incubation medium. The addition of ATP in the presence of NADPH also caused a significant enhancement of the production of O2 radicals. These effects of ATP were not observed with membranes isolated from untreated neutrophils. The release of the serine proteinase is almost fully dependent on the addition of ATP and is correlated with the phosphorylation of membrane proteins. It is also markedly inhibited by the addition of retinal or trifluoperazine inhibitors of native protein kinase C. The results represent the first direct demonstration of a role for membrane-bound protein kinase C activity in the release of neutral proteinase and the production of O2 radicals, responses related to the cytotoxic effects of activated neutrophils.     

Mitochondrial reactive oxygen species in mice lacking superoxide dismutase 2: attenuation via antioxidant treatment

Mice that lack the mitochondrial form of superoxide dismutase (SOD2) incur severe pathologies and mitochondrial deficiencies, including major depletion of complex II, as a consequence of buildup of endogenous reactive oxygen species (Melov, S., Coskun, P., Patel, M., Tuinstra, R., Cottrell, B., Jun, A. S., Zastawny, T. H., Dizdaroglu, M., Goodman, S. I., Huang, T. T., Miziorko, H., Epstein, C. J., and Wallace, D. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 846-851 and Li, Y., Huang, T. T., Carlson, E. J., Melov, S., Ursell, P. C., Olson, J. L., Noble, L. J., Yoshimura, M. P., Berger, C., Chan, P. H., Wallace, D. C., and Epstein, C. J. (1995) Nat. Genet. 11, 376-381). These problems can be greatly attenuated or rescued by synthetic antioxidant treatment, such as with the catalytic antioxidant EUK189 (Hinerfeld, D., Traini, M. D., Weinberger, R. P., Cochran, B., Doctrow, S. R., Harry, J., and Melov, S. (2004) J. Neurochem. 88, 657-667). We have used heart mitochondria from sod2 null mice to better understand mitochondrial reactive oxygen species production both in the absence of SOD2 and following in vivo antioxidant treatment. Isolated heart mitochondria from 5-day-old sod2 null animals respiring on the complex II substrate succinate exhibited statistically significant higher levels of mitochondrial O2* (157%, p < 0.01) but significantly less H2O2 (33%, p < 0.001) than wild type littermates. Treatment of sod2 nullizygous mice with EUK189 proportionately increased the levels of complex II and H2O2. Increased production of O2* resulting from complex II normalization had no effect on steady state levels due to the rapid conversion to H2O2, a process presumably aided by the presence of the EUK189, an SOD mimetic.     

Book Reviews

Books reviewed in this article:
BERNARDO, J. M., BERGER, J. O., DAWID, A. P., and SMITH, A. F. M. (editors). Bayesian Statistics 6.
VAN DER VAART, A. W. Asymptotic Statistics.
NOLAN, D. and SPEED, T. StatLabs: Mathematical Statistics Through Applications.
BINNS, M. R., NYROP, J. P., and VAN DER WERF, W. Sampling and Monitoring in Crop Protection: The Theoretical Basis for Developing Practical Decision Guides.
DIECKMANN, U., LAW, R., and METZ, J. A. J. (editors). The Geometry of Ecological Interactions: Simplifying Spatial Complexity.
LAWSON, A., BIGGERI, A., BOHNING, D., LESAFFRE, E., VIEL, 3.-F., and BERTOLLINI, R. Disease Mapping and Risk Assessment for Public Health.
BLAND, M. and PEACOCK, J. Statistical Questions in Evidence Based Medicine. Oxford University Press, Oxford, 2000.
MANLY, B. F. J. Statistics for Environmental Science and Management.
RÍOS INSUA, D. and RUGGERI, F. (editors) Robust Bayesian Analysis.
BARNDORFF-NIELSEN, 0. E., COX, D. R., and KLUP-PELBERG, C. Complex Stochastic Systems.
PETRIE, A. and SABIN, C. Medical Statistics at a Glance.
THERNEAU, T. M. and GRAMBSCH, P. M. Modeling Survival Data: Extending the Cox Model.
TAN, W.-Y. Stochastic Modeling of AIDS Epidemiology and HIV Pathogenesis.
CHATFIELD, C. Time-Series Forecasting.
MATTHEWS, J. N. S. An Introduction to Randomized Controlled Clinical Trials.
CLARK, I. and HARPER, W. V. Practical Geostatistics 2000.
TANIGUCHI, M. and KAKIZAWA, Y. Asymptotic Theory of Statistical Inference for Time Series.
KARIAN, Z. A. and DUDEWICZ, E. J. Fitting Statistical Distributions: The Generalized Lambda Distribution and Generalized Bootstrap Methods.     

Book Reviews

Book Reviwed in this article:
Dugger, W. M. and S. Bartnicki-Garcia (Eds.), Structure, Function, and Biosynthesis of Plant Cell Walls.
Brandenburger, W., Parasitische Pilze an Gefäßpflanzen in Europa.
Collins, C., and P. M. Lyne, Microbiological Methods
Heitefuß, R. (unter Mitarbeit von W. O tto -H unze und E. B lum ), NKU-Pflanzenschutz im Ackerbau.     

Amino acid sequence and post-translational modification of stem cell factor isolated from buffalo rat liver cell-conditioned medium.

Stem cell factor (SCF) isolated from culture medium conditioned by Buffalo rat liver cells was subjected to detailed structural analysis. Attempts at direct N-terminal sequencing of the factor indicated that its N terminus is blocked as pyroglutamic acid (Zsebo, K. M., Wypych, J., McNiece, I. K., Lu, H. S., Smith, K. A., Karkare, S. B., Sachdev, R. K., Yuschenkoff, V. N., Birkett, N. C., Williams, L. R., Satyagal, V. N., Bosselman, R. A., Mendiaz, E. A., and Langley, K. E. (1990) Cell 63, 195-201). The removal of the blocking pyroglutamate by pyroglutamate aminopeptidase allowed sequencing of the polypeptide chain to position 47. Stem cell factor was also digested with CNBr, trypsin, Staphylococcus aureus protease (strain V8), and AspN peptidase to generate different sets of peptides that were then separated by reverse-phase high-performance liquid chromatography and sequenced. Sequence of an internal peptide fragment obtained by cleavage of stem cell factor at a single tryptophanyl peptide bond was also obtained. From these analyses, the complete amino acid sequence could be constructed. The factor as isolated is a single polypeptide of 164 or 165 amino acids. The sequence is confirmatory to a sequence deduced from a cDNA sequence and provides important evidence for C-terminal processing of the polypeptide encoded by cDNA. There are four potential N-linked glycosylation sites. Asn65, Asn72, Asn109, and Asn120. Sequence determination of isolated peptides suggested that Asn120 is glycosylated, Asn65 and Asn109 glycosylated in some molecules but not in others, and Asn72 not glycosylated. Amino acids at three positions, i.e. 142, 143, and 155, could not be detected during sequence analysis. Since the gene sequence codes for Ser, Thr, and Thr at these positions (Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S., Ting, J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen, F. W., Mendiaz, E. A., Birkett, N. C., Smith, K. C., Johnson, M. J., Parker, V. P., Flores, J. C., Patel, A. C., Fisher, E. F., Erjavec, H. O., Herrera, C. J., Wypych, J., Sachdev, R. K., Pope, J. A., Leslie, I., Wen, D., Lin, C. W., Cupples, R. L., and Zsebo, K. M. (1990) Cell 63, 203-211), they could be sites of O-linked carbohydrate attachment. The four cysteines form two intramolecular disulfide bonds, Cys4-Cys89 and Cys43-Cys138.     

Implications of the reactive thiol and the proximal non-proline cis-peptide bond in the Structure and function of Vibrio harveyi luciferase

The role of a highly reactive cysteine residue, Cys106, in Vibrio harveyi luciferase in modulating the substrate-enzyme interactions and in turn affecting the enzyme activity has been extensively investigated over the past three decades. Replacing Cys106 with valine dramatically hinders the ability of luciferase to stabilize the C4a-hydroperoxyflavin intermediate [Abu-Soud, H. M., Clark, A. C., Francisco, W. A., Baldwin, T. O., and Raushel, F. M. (1993) J. Biol. Chem. 268, 7699-7706] and consume aldehyde substrate [Xi, L., Cho, K.-W., Herndon, M. E., and Tu, S.-C. (1990) J. Biol. Chem. 265, 4200-4203], therefore markedly decreasing enzyme activity. On the basis of the structure-activity relationship of flavin analogues and the location of the phosphate binding site of flavin mononucleotide (FMN) coupled with molecular modeling, the functional part of the isoalloxazine ring of FMN, the thiol side chain of Cys106, the methyl group of Ala75, and the unique non-prolyl cis-peptide bond between Ala74 and Ala75 were found to be closely packed [Lin, L. Y., Sulea, T., Szittner, R., Vassilyev, V., Purisima, E. O., and Meighen, E. A. (2001) Protein Sci. 10, 1563-1571]. Here, by mutating Ala75 to Gly, we restored key wild-type properties to the C106V mutant, in particular, high enzyme activity and a stable C4a-hydroperoxyflavin intermediate, demonstrating that the primary reason for the dark phenotype of the C106V mutant was the unfavorable steric interaction between Val106 and Ala75 side chains, which could in turn disturb the cis-oriented amide linkage of Ala74 and Ala75. Moreover, significant red shifts in light emission of 3-10 nm were measured for luciferases carrying Val106 with the spectrum of the double mutant C106V/A75G now red shifted to that of Photobacterium phosphoreum luciferase, which also has Val and Gly at positions 106 and 75, respectively. These results strengthen the validity of the binding geometry of the modeled flavin with the re-face of the pyrimidine end of the isoalloxazine ring next to Cys106 and implicate the Ala74-Ala75 cis-peptide as a key component in the bioluminescence reaction.     

Quantitative reevaluation of the redox active sites of crystalline bovine heart cytochrome c oxidase

Approximately 30% of the iron contained in a bovine heart cytochrome c oxidase preparation was removed by crystallization, giving a molecular extinction coefficient 1.25-1.4 times higher than those reported thus far. Six electron equivalents provided by dithionite were required for complete reduction of the crystalline cytochrome c oxidase preparation. The fully reduced enzyme was oxidized with 4 oxidation equivalents provided by molecular oxygen, giving an absorption spectrum slightly, but significantly, different from that of the original fully oxidized form. Four electron equivalents were required for complete reduction of the O(2)-oxidized enzyme. The O(2)-oxidized form, when exposed to excess amounts of O(2), was converted to the original oxidized form which required 6 electrons for complete reduction. A slow reduction of the O(2)-oxidized form without any external reductant added indicates the existence of internal electron donors for heme irons in the enzyme. These results suggest that the 2 extra oxidation equivalents in the original oxidized form, compared with the O(2)-oxidized form, are due to a bound peroxide produced by O(2) and electrons from the internal donors, consistently with a peroxide at the O(2) reduction site in the crystal structure of the enzyme (Yoshikawa, S., Shinzawa-Itoh, K. , Nakashima, R., Yaono, R., Yamashita, E., Inoue, N., Yao, M., Fei, M. J., Peters Libeu, C., Mizushima, T., Yamaguchi, H., Tomizaki, T., and Tsukihara, T. (1998) Science 280, 1723-1729).     

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.     

Interaction of hemojuvelin with neogenin results in iron accumulation in human embryonic kidney 293 cells

Type 2 hereditary hemochromatosis (HH) or juvenile hemochromatosis is an early onset, genetically heterogeneous, autosomal recessive disorder of iron overload. Type 2A HH is caused by mutations in the recently cloned hemojuvelin gene (HJV; also called HFE2) (Papanikolaou, G., Samuels, M. E., Ludwig, E. H., MacDonald, M. L., Franchini, P. L., Dube, M. P., Andres, L., MacFarlane, J., Sakellaropoulos, N., Politou, M., Nemeth, E., Thompson, J., Risler, J. K., Zaborowska, C., Babakaiff, R., Radomski, C. C., Pape, T. D., Davidas, O., Christakis, J., Brissot, P., Lockitch, G., Ganz, T., Hayden, M. R., and Goldberg, Y. P. (2004) Nat. Genet. 36, 77-82), whereas Type 2B HH is caused by mutations in hepcidin. HJV is highly expressed in both skeletal muscle and liver. Mutations in HJV are implicated in the majority of diagnosed juvenile hemochromatosis patients. In this study, we stably transfected HJV cDNA into human embryonic kidney 293 cells and characterized the processing of HJV and its effect on iron homeostasis. Our results indicate that HJV is a glycosylphosphatidylinositol-linked protein and undergoes a partial autocatalytic cleavage during its intracellular processing. HJV co-immunoprecipitated with neogenin, a receptor involved in a variety of cellular signaling processes. It did not interact with the closely related receptor DCC (deleted in Colon Cancer). In addition, the HJV G320V mutant implicated in Type 2A HH did not co-immunoprecipitate with neogenin. Immunoblot analysis of ferritin levels and transferrin-55Fe accumulation studies indicated that the HJV-induced increase in intracellular iron levels in human embryonic kidney 293 cells is dependent on the presence of neogenin in the cells, thus linking these two proteins to intracellular iron homeostasis.     

Synaptic vesicle recycling in synapsin I knock-out mice

The synapsins are a family of four neuron-specific phosphoproteins that have been implicated in the regulation of neurotransmitter release. Nevertheless, knock-out mice lacking synapsin Ia and Ib, family members that are major substrates for cAMP and Ca2+/ Calmodulin (CaM)-dependent protein kinases, show limited phenotypic changes when analyzed electrophysiologically (Rosahl, T.W., D. Spillane, M. Missler, J. Herz, D.K. Selig, J.R. Wolff, R.E. Hammer, R.C. Malenka, and T.C. Sudhof. 1995. Nature (Lond.). 375: 488-493; Rosahl, T.W., M. Geppert, D. Spillane, D., J. Herz, R.E. Hammer, R.C. Malenka, and T.C. Sudhof. 1993. Cell. 75:661-670; Li, L., L.S. Chin, O. Shupliakov, L. Brodin, T.S. Sihra, O. Hvalby, V. Jensen, D. Zheng, J.O. McNamara, P. Greengard, and P. Andersen. 1995. Proc. Natl. Acad. Sci. USA. 92:9235- 9239; see also Pieribone, V.A., O. Shupliakov, L. Brodin, S. Hilfiker- Rothenfluh, A.J. Czernik, and P. Greengard. 1995. Nature (Lond.). 375:493-497). Here, using the optical tracer FM 1-43, we characterize the details of synaptic vesicle recycling at individual synaptic boutons in hippocampal cell cultures derived from mice lacking synapsin I or wild-type equivalents. These studies show that both the number of vesicles exocytosed during brief action potential trains and the total recycling vesicle pool are significantly reduced in the synapsin I- deficient mice, while the kinetics of endocytosis and synaptic vesicle repriming appear normal.     

Buchbesprechungen / Book Reviews

Book reviewed in this article:
Sachs, L. , Angewandte Statistik (Anwendung statistischer Methoden).
Progress in Botany (Fortschritt der Botanik). Morphology—Physiology—Genetics—Taxonomy—Geobotany. Herausgeber: K. E sser , K. K ubitzki , M. R unge , E. S chnepf , H. Z iegler .
Cummins, G. B., and Yasuyuki Hiratsuka , Illustrated Genera of Rust Fungi.
Pirson, A., and M. H. Zimmermann (Eds.) , Encyclopedia of Plant Physiology, New Series, Vol 12 D. O. L. L ange , P. S. N obel , C. B. O smond , H. Z ieg - ler (Eds.): Physiological Plant Ecology IV.
Mass, J. L. (ed) , Compendium of Strawberry Diseases.     

Ligand recombination to the alpha and beta subunits of human hemoglobin

The rebinding of CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide to isolated alpha and beta chains and intact hemoglobin at pH 7, 20 degrees C was examined both during and after a 30-ns dye laser pulse. The resultant absorbance changes were analyzed in terms of a linear three-step reaction scheme: Hb + X in equilibrium with C in equilibrium with B in equilibrium with A or HbX, where A is the final bound state, and C and B are geminate states. Rate constants were assigned for each of the transitions in this mechanism using fitting procedures described previously for analyzing ligand rebinding to sperm whale myoglobin at room temperature (Gibson, Q. H., Olson, J. S., McKinnie, R. E., and Rohlfs, R. J. (1986) J. Biol. Chem. 261, 10228-10239). Five major conclusions were obtained. First, initial geminate recombination phases for the NO and O2 complexes of hemoglobin and its isolated subunits exhibit half-times equal to approximately 12 and approximately 440 ps, respectively. These values are in excellent agreement with more direct, picosecond measurements of the geminate recombination of HbNO (Cornelius, P. A., Hochstrasser, R. M., and Steele, A. W. (1983) J. Mol. Biol. 163, 119-128) and HbO2 (Friedman, J. M., Scott, T. W., Fisanick, G. J., Simon, S. R., Findsen, E. W., Ondrias, M. R., and MacDonald, V. W. (1985) Science 229, 187-229) following extremely short laser pulses. Second, the correspondence between our nanosecond measurements and the published picosecond data suggests strongly that the intrinsic photochemical yield of all ferrous, hexacoordinate heme complexes approaches one. Third, the major differences between the isolated alpha and beta chains involve the rate of ligand migration to the solvent, kC----X and the extent of recombination from the second geminate state, C, as measured by the ratio kC----B/kC----X. Fourth, for both isolated chains and intact hemoglobin, the rate and equilibrium constants for the formation of the initial O2 geminate state starting from ligand in the solvent (i.e. kX----B and KX----B) are 5-10 times greater than the corresponding parameters for the formation of the first CO geminate state. Fifth, the rate-limiting step for NO, O2, and isonitrile binding to hemoglobin and its isolated subunits is ligand migration up to the initial geminate state (i.e. kX----B). In the case of CO binding, both migration to state B and iron-ligand bond formation (kB----A) affect the overall, bimolecular association rate constant.     

Books

Book reviews in this article:
A ntarcitc E cology . R. M. Laws (ed.).
S eals O f T he W orld . J. E. King.
R eproduction I n W hales , D olphins and P orpoises . W. F. Perrin, R. L. Brownell, Jr., and D. P. DeMaster, eds.
E volution I n T he G alapagos I slands . R. J. Berry (ed.).
H istorical W haling R ecords . M. F. Tillman and G. P. Donovan (eds.).
D iving and A sphyxia , A C omparative S tudy O f A nimals and M an . R. Elsner and B. Gooden.