Relative susceptibilities of the interchain disulfides of an immunoglobulin G molecule to reduction by dithiothreitol.

The reduction by dithiothreitol (DTT) of the four interchain disulfides of a human IgGlkappa immunoglobulin has been studied by two methods: variation of the concentration of DTT relative to the protein concentration (incremental reduction); and variation of the time of reduction at fixed levels of DTT and protein (kinetic reduction). In both cases, the results depend on whether the reduction is carried out aerobically or anaerobically. Under aerobic conditions, the relative levels of intermediates (HL, H2, and H2L) which are generated as native molecules (H2L2) are converted to reduced heavy (H) and light (L) chains depend on the concentrations of protein and DTT as well as on the exposure time to DTT; no stable equilibrium is reached between reduced and oxidized states and conditions gradually revert from those favoring reduction to those favoring reoxidation. By contrast, anaerobic reduction is independent of protein concentration or time of exposure to DTT, beyond about 30 min, indicating that an equilibrium between partially reduced and oxidized states is achieved. The distribution of intermediates observed under anaerobic conditions has been analyzed according to theoretical models (Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (second in a series of three articles in this issue)). Within experimental error, both kinds of anaerobic experiments resemble a random reduction process wherein the four disulfides are equivalent and independent of each other with respect to rate and extent of reduction by D. It is concluded that there are no readily detected pathways in the process, as would occur if the intrinsic reactivities of the bonds were distinct, and no marked cooperatively between the four reaction sites, as would be observed if reduction of one bond materially facilitated or hampered reactivity at another site. Both of these characteristics of the reduction are in direct contrast to those of the reoxidative process, which is marked by the initial preference for formation of a bond between heavy and light chains, and by kinetic cooperativity in bond formation during the course of the reaction (Sears, D.W., et al. (1977), Biochemistry 16 (first in a series of three articles in this issue); Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (second in this series)).     

Acquisition of the covalent quaternary structure of an immunoglobulin G molecule. Reoxidative assembly in vitro.

We recently reported results of an investigation of the reoxidation of a human, monoclonal immunoglobulin G, following selective reduction of its interchain disulfides by dithiothreitol (Sears, D.W., et al. (1975), Proc. Natl. Acad. Sci. U.S.A. 72, 353). In that work, we described the reoxidative behavior of the molecule under nondissociating conditions. In the present paper, results are presented of the reoxidation of heavy (H) and light (L) chains of this protein alone, or mixed in varying proportions after separation, or mixed with the L chains modified prior to recombination and reoxidation. The overall reoxidative asembly patterns in experiments with H and L separated prior to recombination are similar to those observed when the chains remain noncovalently associated throughout. With equimolar mixtures of H and L, the reoxidation rates also are similar to those of unseparated chains. However, when L chains are present in excess, the overall in vitro rates of covalent assembly are generally diminished, probably indicating transient nonproductive interactions. At the highest molar excesses of L (3:1), the assembly pathways may also be modified. In all experiments with excess L chains, covalent L2 dimers form at rates which are comparatively slow relative to the H2L2 assembly rates. Two kinds of reoxidation experiments with modified L chains are described here for the first time. In the first, the free half-cystine of L is irreversibly blocked by reaction with iodoacetamide, and the alkylated L chains are recombined with reduced H chains. This experiment isolates the reactions in which H2 disulfides are formed without the accompanying formation of HL bonds. Although the alkylated L chains do not play a direct role in the reoxidation, their presence is required to inhibit aggregation and precipitation of high-molecular-weight products which otherwise ensue; this suggests a possible biological role for excess L in vivo. In the second kind of experiment, covalent L2 dimers are mixed with reduced H chains. L2 rapidly disappears with the concurrent appearance of HL, H2L, and fully assembled H2L. H2 dimers are also reactive in this process. Special procedures were developed for analyzing the data from these experiments. A complete format is given for the quantitative determination of the concentration of each of the molecular components directly from spectroscopic scans of the gels. The computational methods solve the general analytical problem posed when staining is not proportional to mass and are applicable to a wide variety of systems utilizing gel electrophoresis to study subunit interactions. A theoretical analysis of pathway and kinetic cooperatively in this system is presented in the following paper (Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (following paper in this issue)).     

Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. Studies with specifically tritiated (2R)-methylmalonyl-CoA as substrate.

(2R)-Methyl[2-3H]malonyl-CoA was used as the substrate for methylmalonyl-CoA epimerase from Propionibacterium shermanii, under conditions where the (2S)-methylmalonyl-CoA product was removed enzymically as fast as it was formed, and the fate of the label was monitored at different extents of reaction. Very little, if any, tritium is found attached to the C-2 position in the (2S)-epimer product (isolated as propionyl-CoA). Evidently, the hydrogen atom of the new C-H bond in the product is essentially solvent-derived. The rate of tritium release into the solvent is lower than the rate of product formation, and shows a primary kinetic tritium-isotope effect on kcat./Km of 2.3 +/- 0.1. The specific radioactivity of the remaining substrate rises slowly during the epimerase-catalysed reaction, and this provides an independent estimate of the primary kinetic tritium-isotope effect on kcat./Km of 1.6 +/- 0.5. These results, taken together, indicate that the mechanistic pathway of the epimerase-catalysed reaction resembles that established for proline racemase [Cardinale & Abeles, (1968) Biochemistry 7, 3970-3978], in which two enzyme bases are involved in catalysis. One base removes the proton from the substrate, the second provides the new proton, and there is no fast isotopic exchange between enzyme-bound intermediates and solvent protons.     

Reoxidation of reduced bovine growth hormone from a stable secondary structure

In order to determine solution conditions appropriate for reoxidizing reduced bovine growth hormone (bGH), we have examined the possibility of using a particular denaturant concentration to poise the secondary and tertiary structure of the reduced protein in a stable, nativelike state. It was envisioned that the structure of the reduced molecule would differ from that of the final oxidized molecule solely by the absence of disulfide bonds. Dilution of concentrated samples of reduced and unfolded protein from 6.0 M guanidine into 4.5 M urea followed by air oxidation indicated it was possible to induce refolding and reoxidation to an oxidized monomeric species in high yield (approximately 90%). The choice of solution conditions was based on comparison of urea equilibrium denaturation data for native oxidized protein to those for completely reduced protein and to protein in which sulfhydryl groups had been either partially or completely reduced and subjected to modification with iodoacetamide or methyl methanethiolsulfonate. The denaturation behavior of these species supports the existence of equilibrium folding intermediates for bovine growth hormone and demonstrates that chemical modification of the protein is capable of inducing differences in the denaturation behavior of these intermediates. The changes in the protein absorption spectrum and helix-related circular dichroism signal, along with direct titration of protein sulfhydryl groups, indicated that the refolding/reoxidation of bGH is a multistate process. The ordered nature of the kinetic changes in these probes during reoxidation indicates that disulfide formation is a sequential process, with little mispairing in 4.5 M urea, and that it proceeds through one or more obligatory kinetic folding events. The equilibrium denaturation behavior of the oxidized molecule and the various chemically modified forms, together with the reoxidation data, indicated that the protein maintains a high degree of secondary structure without intrachain disulfide bonds. The formation of these disulfide bonds is a discrete process which occurs after a framework of protein secondary structure is established.     

Differential scanning calorimetric study of the effect of sterol side chain length and structure on dipalmitoylphosphatidylcholine thermotropic phase behavior.

We have investigated the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers containing a series of cholesterol analogues varying in the length and structure of their alkyl side chains. We find that upon the incorporation of up to approximately 25 mol % of any of the side chain analogues, the DPPC main transition endotherm consists of superimposed sharp and broad components representing the hydrocarbon chain melting of sterol-poor and sterol-rich phospholipid domains, respectively. Moreover, the behavior of these components is dependent on sterol side chain length. Specifically, for all sterol/DPPC mixtures, the sharp component enthalpy decreases linearly to zero by 25 mol % sterol while the cooperativity is only moderately reduced from that observed in the pure phospholipid. In addition, the sharp component transition temperature decreases for all sterol/DPPC mixtures; however, the magnitude of the decrease is dependent on the sterol side chain length. With respect to the broad component, the enthalpy initially increases to a maximum around 25 mol % sterol, thereafter decreasing toward zero by 50 mol % sterol with the exception of the sterols with very short alkyl side chains. Both the transition temperature and cooperativity of the broad component clearly exhibit alkyl chain length-dependent effects, with both the transition temperature and cooperativity decreasing more dramatically for sterols with progressively shorter side chains. We ascribe the chain length-dependent effects on transition temperature and cooperativity to the hydrophobic mismatch between the sterol and the host DPPC bilayer (see McMullen, T. P. W., Lewis, R. N. A. H., and McElhaney, R. N. (1993) Biochemistry 32:516-522).(ABSTRACT TRUNCATED AT 250 WORDS)     

Crotonase-catalyzed beta-elimination is concerted: a double isotope effect study

Determining the sequence of bond cleavages, and consequently the nature of intermediates, in enzyme-catalyzed reactions is a major goal of mechanistic enzymology. When significant primary isotope effects on V/K are observed for two different bond cleavages, both bonds may be broken in the same transition state or they can reflect two different transition states that are of nearly identical energy and consequently both are partially rate limiting. For the crotonase-catalyzed dehydration of 3-hydroxybutyrylpantetheine, the primary D(V/K) and 18(V/K) are 1.60 and 1.053 [Bahnson, B. J., & Anderson, V. E. (1989) Biochemistry 28, 4173-4181], respectively. In this case, double isotope effects can discriminate between the two possibilities [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106-5114; Belasco, J. G., Albery, W. J., & Knowles, J. R. (1983) J. Am. Chem. Soc. 105, 2475-2477]. The ratio of the alpha-secondary D(V/K) for the hydration of crotonylpantetheine catalyzed by crotonase in H2O and D2O has been determined to be 1.003 +/- 0.006. The invariance of the alpha-secondary effect where the chemical reaction is completely rate determining requires that both bond cleavages be concerted or that the substitution of 2H at the primary position not significantly alter the partitioning of a hypothetical carbanion. The observation of a solvent discrimination isotope effect determined from the relative incorporation of 2H from 50% D2O of 1.60 +/- 0.03, identical with the primary D(V/K), and the determination that the rate of exchange of the abstracted proton with solvent proceeds at less than 3% of the overall reaction rate also fail to provide evidence for a carbanion intermediate and are consistent with a concerted reaction. Identical primary D(V/K)s determined in H2O and D2O indicate that there is not a significant solvent isotope effect on C-O bond cleavage. The isotope ratios determined in these studies were performed by negative ion chemical ionization whole molecule mass spectrometry of the pentafluorobenzyl esters, a new method whose validity is established by comparison with previously determined kinetic and equilibrium isotope effects.     

Mouse immunoglobulin mu heavy chain mRNA of Y5781, a high yield myeloma.

The BALB/c myeloma tumor, Y5781, has a high level of mu heavy chain mRNA and kappa light chain mRNA, as suggested by denaturing gel analyses of poly(A)-rich, total polysomal mRNA, and confirmed for the mu heavy chain mRNA by kinetic complexity analyses. Both the mRNA coding for the heavy and light chains appear as very prominent and discrete peaks above the generally polydisperse background of the total polysomal mRNA. This mRNA level appears to be stable through a limited number of subcutaneous passages of this myeloma, providing a potentially useful system for mu heavy chain mRNA synthesis and processing. The mu heavy chain mRNA of this myeloma has been enriched to about 60% homogeneity by physicochemical means. In agreement with a previous report (Faust, C.H., Jr., Heim, I., and Moore, J. (1979) Biochemistry 18, 1106-1119), the following physical and biological properties were observed. The mature cytoplasmic mu heavy chain mRNA is 950,000 daltons, i.e. about 2800 nucleotides, and contains approximately 800 undefined, nontranslated bases. In an mRNA-dependent cell-free system, this mRNA stimulates the synthesis of a single, serologically reactive mu heavy chain-like protein, confirmed by tryptic peptide maps.     

Early intermediates in the folding of dihydrofolate reductase from Escherichia coli detected by hydrogen exchange and NMR.

The kinetic folding mechanism for Escherichia coli dihydrofolate reductase postulates two distinct types of transient intermediates. The first forms within 5 ms and has substantial secondary structure but little stability. The second is a set of four species that appear over the course of several hundred milliseconds and have secondary structure, specific tertiary structure, and significant stability (Jennings PA, Finn BE, Jones BE, Matthews CR, 1993, Biochemistry 32:3783-3789). Pulse labeling hydrogen exchange experiments were performed to determine the specific amide hydrogens in alpha-helices and beta-strands that become protected from exchange through the formation of stable hydrogen bonds during this time period. A significant degree of protection was observed for two subsets of the amide hydrogens within the dead time of this experiment (6 ms). The side chains of one subset form a continuous nonpolar strip linking six of the eight strands in the beta-sheet. The other subset corresponds to a nonpolar cluster on the opposite face of the sheet and links three of the strands and two alpha-helices. Taken together, these data demonstrate that the complex strand topology of this eight-stranded sheet can be formed correctly within 6 ms. Measurement of the protection factors at three different folding times (13 ms, 141 ms, and 500 ms) indicates that, of the 13 amide hydrogens displaying significant protection within 6 ms, 8 exhibit an increase in their protection factors from approximately 5 to approximately 50 over this time range; the remaining five exhibit protection factors > 100 at 13 ms. Only approximately half of the population of molecules form this set of stable hydrogen bonds. Thirteen additional hydrogens in the beta-sheet become protected from exchange as the set of native conformers appear, suggesting that the stabilization of this network reflects the global cooperativity of the folding reaction.     

Stability and substructure of cardiac myosin subfragment 1 and isolation and properties of its heavy-chain subunit

The substructure and the thermal stability of the subunit interactions of bovine cardiac myosin subfragment 1 (SF1) have been examined. The results are in agreement with previous reports that the cardiac protein is cleaved in a very similar manner [Flink, I. L., & Morkin, E. (1982) Biophys. J. 37, 34; Korner, M., Thiem, N. V., Cardinaud, R., & Lacombe, G. (1983) Biochemistry 22, 5843-5847] but at a much faster rate [Applegate, D., Azarcon, A., & Reisler, E. (1984) Biochemistry 23, 6626-6630] than the skeletal protein. Additionally, it is found that the long-lived, steady-state intermediates formed by these proteins with MgATP at high ionic strength differ in their susceptibilities to tryptic attack especially at the 27K/50K junction of the associated heavy chains, suggesting a different conformation for these intermediates of the cardiac and skeletal SF1's. The thermal stability of the subunit interactions under conditions approaching the physiological state was examined by thermal ion-exchange chromatography of cardiac SF1 at 39.5 degrees C in the presence of MgATP. This results in the separation of part of the protein as the isolated heavy chain which is found to exhibit high levels of ATPase activity in the absence and presence of actin. Tryptic digestion of cardiac SF1 prior to thermal ion-exchange chromatography produces greater dissociation, with the heavy chain in this case being isolated as a complex of 27K, 50K, and 18-20K fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

The catalytic mechanism of EPSP synthase revisited.

Recent analysis of EPSP synthase by solid-state NMR has led to the postulation of a new enzyme reaction pathway and raised once again the question of an intermediate species covalently bound to the enzyme [Studelska, D., McDowell, L., Espe, M., Klug, C., and Schaefer, J. (1997) Biochemistry 36, 15555-15560]. Therefore, we have reexamined the mechanism of the reaction catalyzed by EPSP synthase and analyzed the reaction products formed under the conditions used in preparing samples for solid-state NMR. Single-turnover experiments were carried out using both [1-14C]- and [32P]PEP showing the formation and decay of the previously proposed tetrahedral intermediate species on a time scale comparable with the disappearance of substrate and formation of product, thus unequivocally establishing the kinetic competence. The possible presence of a covalently bound enzyme intermediate species was also investigated, using SDS-PAGE and Centricon concentration analysis of the quenched reaction samples. No covalently bound enzyme intermediates were observed during the reaction. An enzyme assay was also performed repeating the conditions used in sample preparation for the solid-state NMR studies. We show that under these conditions, total turnover of substrates to products was observed within 45 s at -30 degrees C prior to freezing and lyophilization. Following lyophilization, the samples were stored at -20 degrees C and analyzed over a period of 21 days. We observed the conversion of the product EPSP into the side product, a cyclic EPSP ketal, and the breakdown product, pyruvate. Thus, the new species reported by solid-state NMR can be accounted for by previously characterized reaction products and side products formed during sample preparation and upon incubation in the solid-state. Our conclusions are also supported by the solution and solid-state NMR studies recently reported [Jakeman et al. (1998) Biochemistry 37, 12012-12019]. These results once again highlight the importance of kinetic competence as a criterion to be used in defining enzyme intermediates and point to the errors in interpretation of results when the time dependence of formation of the proposed intermediates is not considered.     

The dissociation of carbon monoxide from hemoglobin intermediate

To investigate the mechanism of allosteric switching in human hemoglobin, we have studied the dissociation of the ligand (CO) from several intermediate ligation states by a stopped-flow kinetic technique that utilizes competitive binding of CO by microperoxidase. The hemoglobin species investigated include Hb(CO)4, the diliganded symmetrical species (alpha beta-CO)2 and (alpha-CO beta)2, and the di- and monoliganded asymmetrical species (alpha-CO beta-CO)(alpha beta), (alpha-CO beta)(alpha beta-CO), (alpha beta-CO) (alpha beta), and (alpha-CO beta)(alpha beta). They were obtained by rapid reduction with dithionite of the corresponding valence intermediates that in turn were obtained by chromatography or by hybridization. The nature and concentration of the intermediates were determined by isoelectric focusing at -25 degrees C. The study was performed at varying hemoglobin concentrations (0.1, 0.02, and 0.001 mM [heme]), pH (6.0, 7.0, 8.0), with and without inositol hexaphosphate. The results indicate that: (a) hemoglobin concentration in the 0.1-0.02 mM range does not significantly affect the kinetic rates; (b) the alpha chains dissociate CO faster than the beta chains; (c) the symmetrical diliganded intermediates show cooperativity with respect to ligand dissociation that disappears in the presence of inositol hexaphosphate; (d) the monoliganded intermediates dissociate CO faster than the diliganded intermediates; (e) the asymmetrical diliganded intermediates are functionally different from the symmetrical species.     

A theoretical study on the hydrolysis mechanism of carbon disulfide

The hydrolysis mechanism of CS(2) was studied using density functional theory. By analyzing the structures of the reactant, transition states, intermediates, and products, it can be concluded that the hydrolysis of CS(2) occurs via two mechanisms, one of which is a two-step mechanism (CS(2) first reacts with an H(2)O, leading to the formation of the intermediate COS, then COS reacts with another H(2)O, resulting in the formation of H(2)S and CO(2)). The other is a one-step mechanism, where CS(2) reacts with two H(2)O molecules continuously, leading to the formation of H(2)S and CO(2). By analyzing the thermodynamics and the change in the kinetic function, it can be concluded that the rate-determining step involves H and OH in H(2)O attacking S and C in CS(2), respectively, causing the C=S double bond to change into a single bond. The two mechanisms are competitive. When performing the hydrolysis of CS(2) with a catalyst, the optimal temperature is below 252°C.     

Hysteresis and positive cooperativity of iceberg lettuce polyphenol oxidase.

A kinetic study of the diphenolase activity of latent polyphenol oxidase (PPO), purified from Iceberg lettuce (Lactuca sativa L), revealed a sigmoid relationship between the reaction rate and the substrate concentration with a high Hill coefficient (n(H) = 3.8). This positive cooperativity had not been previously described for any PPO. Furthermore, the enzyme showed a lag phase in the expression of this activity, suggesting a hysteretic nature of the enzyme. The kinetic behavior, the latency and the lag phase varied at different steps of the purification process. PPO showed hyperbolic or cooperative kinetics depending on the pH assay and the sodium dodecyl sulfate (SDS) concentration. Substrate-induced slow conformational change of the oligomeric enzyme is suggested. The conformational change would be toward a more active enzyme form with higher affinity for the substrate and favoured by acid pH and SDS.     

Catalytic and allosteric mechanism of AMP nucleosidase from primary, beta-secondary, and multiple heavy atom kinetic isotope effects

Adenosine 5'-phosphate was synthesized with specific heavy atom substitutions to permit measurement of V/K kinetic isotope effects for the N-glycohydrolase activity of the allosteric AMP nucleosidase and the acid-catalyzed solvolysis of these compounds. The effects of allosteric activation on the kinetic isotope effects together with the kinetic mechanism of AMP nucleosidase [DeWolf, W. E., Jr., Emig, F. A., & Schramm, V. L. (1986) Biochemistry 25, 4132-4140] indicate that the kinetic isotope effects are fully expressed. Comparison of individual primary and secondary kinetic isotope effects with combined isotope effects and the isotope effect of the reverse reaction indicated that kinetic isotope effects in AMP nucleosidase arise from a single step in the reaction mechanism. Under these conditions, kinetic isotope effects can be used to interpret transition-state structure for AMP nucleosidase. Changes in kinetic isotope effects occurred as a function of allosteric activator, demonstrating that allosteric activation alters transition-state structure for AMP nucleosidase. Kinetic isotope effects, expressed as [V/K(normal isotope]/[V/K(heavy isotope)], were observed with [2'-2H]AMP (1.061 +/- 0.002), [9-15N]AMP (1.030 +/- 0.003), [1'-2H]AMP (1.045 +/- 0.002), and [1'-14C]AMP (1.035 +/- 0.002) when hydrolyzed by AMP nucleosidase in the absence of MgATP. Addition of MgATP altered the [2'-2H]AMP effect (1.043 +/- 0.002) and the [1'-2H]AMP effect (1.030 +/- 0.003) and caused a smaller decrease of the 14C and 15N effects. Multiple heavy atom substitutions into AMP caused an increase in observed isotope effects to 1.084 +/- 0.004 for [1'-2H,1'-14C]AMP and to 1.058 +/- 0.002 for [9-15N,1'-14C]AMP with the enzyme in the absence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Allosteric properties, substrate specificity, and subsite affinities of honeybee alpha-glucosidase I

The substrate specificity of honeybee alpha-glucosidase I, a monomeric enzyme was kinetically investigated. Unusual kinetic features were observed in the cleavage reactions of sucrose, maltose, p-nitrophenyl alpha-glucoside, phenyl alpha-glucoside, turanose, and maltodextrin (DP = 13). At relatively high substrate concentrations, the velocities of liberation of fructose from sucrose, glucose from maltose, p-nitrophenol from p-nitrophenyl alpha-glucoside, and phenol from phenyl alpha-glucoside were accelerated, and so the Lineweaver-Burk plots were convex, indicating negative kinetic cooperativity: the Hill coefficients were calculated to be 0.50, 0.64, 0.50, and 0.67 for sucrose, maltose, p-nitrophenyl alpha-glucoside, and phenyl alpha-glucoside, respectively. For the degradation of turanose and maltodextrin, the enzyme showed a sigmoidal curve in v versus s plots and thus catalyzed the reaction with positive kinetic cooperativity. The Lineweaver-Burk plots were concave and the Hill coefficients were 1.2 and 1.5 for turanose and maltodextrin, respectively. These unique properties cannot be interpreted by the reaction mechanism that Huber and Thompson proposed: (1973) Biochemistry 12, 4011-4020. The rate parameters for the hydrolysis of sucrose, maltose, p-nitrophenyl alpha-glucoside and phenyl alpha-glucoside were estimated by extrapolating the linear part of the Lineweaver-Burk plots at low substrate concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural control of cytochrome P450-catalyzed ω-hydroxylation

The regiospecific or preferential ω-hydroxylation of hydrocarbon chains is thermodynamically disfavored because the ease of C–H bond hydroxylation depends on the bond strength, and the primary C–H bond of a terminal methyl group is stronger than the secondary or tertiary C–H bond adjacent to it. The hydroxylation reaction will therefore occur primarily at the adjacent secondary or tertiary C–H bond unless the protein structure specifically enforces primary C–H bond oxidation. Here we review the classes of enzymes that catalyze ω-hydroxylation and our current understanding of the structural features that promote the ω-hydroxylation of unbranched and methyl-branched hydrocarbon chains. The evidence indicates that steric constraints are used to favor reaction at the ω-site rather than at the more reactive (ω−1)-site.     

The oxygen reactions of reduced cytochrome c oxidase. Position of a form with an unusual EPR signal in the sequence of early intermediates.

The order of appearance of intermediates in the reoxidation of reduced cytochrome c oxidase by oxygen has been examined. Particular emphasis was placed on determining where the intermediate with the EPR signal at g = 5, 1.78, 1.69 (Shaw, R.W., Hansen, R.E. and Beinert, H. (1978) J. Biol. Chem. 253, 6637--6640) appears in the sequence of events during reoxidation. Flash photolysis of reduced, CO-complexed samples of cytochrome c oxidase in the presence of oxygen in a buffer containing 30% (v/v) ethylene glycol at 77 K and 195 K has been used to generate states of partial reoxidation. The intermediate with the EPR signal at g = 5, 1.78, and 1.69 can be detected as a product of the photolysis and subsequent oxidation but does not appear until the photolyzed sample is incubated at temperatures well above 196 K. In the course of the reoxidation, the intermediate characterized by the g = 5, 1.78, 1.69 signal occurs in the reaction sequence after the states referred to as 'Compound A' and 'Compound B' (Chance, B., Saronio, C., and Leigh, J.S. (1975) J. Biol. Chem. 250, 9226--9237). Its appearance is within the time range reported for the formation of 'oxygenated' cytochrome c oxidase (Orii, Y. (1979) in Cytochrome Oxidase (King, T.E., Orii, Y., Chance, B. and Okunuki, K., eds.), pp. 331--340, Elsevier/North-Holland Biomedical Press, Amsterdam).     

Studies on the alkali light chains of vertebrate skeletal muscle myosin. Effect of tyrosyl modification on the ability to reassociate to heavy chains

The structures of the alkali light chain subunits A1 and A2 have been studied by examining the effect of the conformationally sensitive reagent tetranitromethane, which reacts specifically with tyrosyl residues. Whereas reaction in the presence of 6 M guanidine hydrochloride results in modification of the three tyrosyl residues of both these light chains, only two tyrosyl residues are exposed to the reagent in the native conformations of these proteins. By gel chromatography of the CNBr-cleaved chains it was demonstrated that the two reactive tyrosyls are those located in the CB-1 and CB-3 segments and that these tyrosyl residues are modified simultaneously and not sequentially. The unreactive tyrosyl residue is in the CB-6 segment and is separated by two residues from the single cysteinyl residue of these chains. It is found that the modified light chains cannot be made to reassociate with the heavy chains by the NH4Cl hybridization procedure of Wagner and Weeds [J. Mol. Biol. 109, 455-470 (1977)] or by the thermal hybridization procedure [Burke and Sivaramakrishnan (1981) Biochemistry 20, 5908-5913]. Furthermore, reduction of the nitrotyrosyl groups to aminotyrosyl residues by sodium dithionite does not restore this effect. The data suggest that regions of the light chains at CB-1 and CB-3 are involved in the association to the heavy chains.