High affinity binding of Bcl-xL to cytochrome c: possible relevance for interception of translocated cytochrome c in apoptosis

The release of cytochrome c from mitochondria and apoptosis relies on several preferential and selective interactions involving the Bcl-2 family of proteins. There is, however, no direct evidence for the interaction of cytochrome c with these proteins at any stage of apoptosis. To investigate if any pro-survival protein from the Bcl-2 family could intercept cytochrome c after its translocation from mitochondria, the interaction of cytochrome c with bacterially expressed human Bcl-x(L) was studied at pH 7. In size-exclusion chromatography, purified full-length His(6)-tagged Bcl-x(L) migrated as both dimer and monomer, of which the monomeric fractions were used for experiments. Coimmunoprecipitation studies show that cytochrome c interacts with Bcl-x(L). The extent of caspase activity in cell lysate elicited by externally added cytochrome c is reduced when a preincubated mixture of Bcl-x(L) and cytochrome c is used instead. Equilibrium binding monitored by optical absorption of cytochrome c as a function of titrating concentrations of Bcl-x(L) yields the association constant, K(ass) = 8.4(+/- 4) x 10(6) M(-1) (binding affinity, K(diss) = 1/K(ass) approximately 120 nM) which decreases at high ionic strength. The rates for binding of Bcl-x(L) to cytochrome c, studied by stopped-flow kinetics at pH 7, show that the bimolecular rate constant for binding, k(bi) = 0.24 x 10(6) M(-1) s(-1). Values of the thermodynamic and kinetic parameters for Bcl-x(L)-cytochrome c interaction are very similar to those known for regulatory protein-protein interactions in apoptosis.     

Cytochrome c peroxidase-cytochrome c complex: locating the second binding domain on cytochrome c peroxidase with site-directed mutagenesis

Cytochrome c peroxidase (CcP) can bind as many as two cytochrome c (Cc) molecules in an electrostatic complex. The location of the two binding domains on CcP has been probed by photoinduced interprotein electron transfer (ET) between zinc-substituted horse cytochrome c (ZnCc) and CcP with surface charge-reversal mutations and by isothermal titration calorimetry (ITC). These results, which are the first experimental evidence for the location of domain 2, indicate that the weak-binding domain includes residues 146-150 on CcP. CcP(E290K) has a charge-reversal mutation in the tight-binding domain, which should weaken binding, and it weakens the 1:1 complex; K(1) decreases 20-fold at 18 mM ionic strength. We have employed two mutations to probe the proposed location for the weak-binding domain on the CcP surface: (i) D148K, a "detrimental" mutation with a net (+2) change in the charge of CcP, and (ii) K149E, a "beneficial" mutation with a net (-2) change in the charge. The interactions between FeCc and CcP(WT and K149E) also have been studied with ITC. The CcP(D148K) mutation causes no substantial change in the 2:1 binding but an increase in the reactivity of the 2:1 complex. The latter can be interpreted as a long-range influence on the heme environment or, more likely, the enhancement of a minority subset of binding conformations with favorable pathways for ET. CcP(K149E) has a charge-reversal mutation in the weak-binding domain that produces a substantial increase in the 2:1 binding constant as measured by both quenching and ITC. For the 1:1 complex of CcP(WT), DeltaG(1) = -8.2 kcal/mol (K(1) = 1.3 x 10(6) M(-)(1)), DeltaH(1) = +2.7 kcal/mol, and DeltaS(1) = +37 cal/K.mol at 293 K; for the second binding stage, K(2) < 5 x 10(3) M(-)(1), but accurate thermodynamic parameters were not obtained. For the 1:1 complex of CcP(K149E), DeltaG(1) = -8.5 kcal/mol (K(1) = 2 x 10(6) M(-)(1)), DeltaH(1) = +2. 0 kcal/mol, and DeltaS(1) = +36 cal/K.mol; for the second stage, DeltaG(2) = -5.5 kcal/mol (K(1) = 1.3 x 10(4) M(-)(1)), DeltaH(2) = +2.9 kcal/mol, and DeltaS(2) = +29 cal/K.mol.     

Interaction of isofraxidin with human serum albumin

This study was designed to examine the interaction of isofraxidin with human serum albumin (HSA) under physiological conditions with drug concentrations in the range of 3.3 x 10(-6) mol L(-1)-3.0x10(-5) mol L(-1) and HSA concentration at 1.5 x 10(-6) mol L(-1). Fluorescence quenching methods in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy were used to determine the drug-binding mode, the binding constant and the protein structure changes in the presence of isofraxidin in aqueous solution. Spectroscopic evidence showed that the interaction results in one type of isofraxidin-HSA complex with binding constants of 4.1266 x 10(5) L mol(-1), 3.8612 x 10(5) L mol(-1), 3.5063 x 10(5) L mol(-1), 3.1241 x 10(5) L mol(-1) at 296 K, 303 K, 310 K, 318 K, respectively. The thermodynamic parameters, enthalpy change (DeltaH) and entropy change (DeltaS) were calculated to be -10.08 kJ mol(-1) and 73.57 J mol(-1) K(-1) according to van't Hoff equation, which indicated that hydrophobic interaction played a main role in the binding of isofraxidin to HSA. The experiment results are nearly in accordance with the calculation results obtained by Silicon Graphics Ocatane2 workstation.     

Global effects of the energetics of coenzyme binding: NADPH controls the protein interaction properties of human cytochrome P450 reductase

The thermodynamics of coenzyme binding to human cytochrome P450 reductase (CPR) and its isolated FAD-binding domain have been studied by isothermal titration calorimetry. Binding of 2',5'-ADP, NADP(+), and H(4)NADP, an isosteric NADPH analogue, is described in terms of the dissociation binding constant (K(d)), the enthalpy (DeltaH(B)) and entropy (TDeltaS(B)) of binding, and the heat capacity change (DeltaC(p)). This systematic approach allowed the effect of coenzyme redox state on binding to CPR to be determined. The recognition and stability of the coenzyme-CPR complex are largely determined by interaction with the adenosine moiety (K(d2)(')(,5)(')(-ADP) = 76 nM), regardless of the redox state of the nicotinamide moiety. Similar heat capacity change (DeltaC(p)) values for 2',5'-ADP (-210 cal mol(-)(1) K(-)(1)), NADP(+) (-230 cal mol(-)(1) K(-)(1)), and H(4)NADP (-220 cal mol(-)(1) K(-)(1)) indicate no significant contribution from the nicotinamide moiety to the binding interaction surface. The coenzyme binding stoichiometry to CPR is 1:1. This result validates a recently proposed one-site kinetic model [Daff, S. (2004) Biochemistry 43, 3929-3932] as opposed to a two-site model previously suggested by us [Gutierrez, A., Lian, L.-Y., Wolf, C. R., Scrutton, N. S., and Roberts, C. G. K. (2001) Biochemistry 40, 1964-1975]. Calorimetric studies in which binding of 2',5'-ADP to CPR (TDeltaS(B) = -13400 +/- 200 cal mol(-)(1), 35 degrees C) was compared with binding of the same ligand to the isolated FAD-binding domain (TDeltaS(B) = -11200 +/- 300 cal mol(-)(1), 35 degrees C) indicate that the number of accessible conformational substates of the protein increases upon 2',5'-ADP binding in the presence of the FMN-binding domain. This pattern was consistently observed along the temperature range that was studied (5-35 degrees C). This contribution of coenzyme binding energy to domain dynamics in CPR agrees with conclusions from previous temperature-jump studies [Gutierrez, A., Paine, M., Wolf, C. R., Scrutton, N. S., and Roberts, G. C. K. (2002) Biochemistry 41, 4626-4637]. A combination of calorimetry and stopped-flow spectrophotometry kinetics experiments showed that this linkage between coenzyme binding energetics and diffusional domain motion impinges directly on the molecular recognition of cytochrome c by CPR. Single-turnover reduction of cytochrome c by CPR (k(max) = 15 s(-)(1), K(d) = 37 microM) is critically coupled to coenzyme binding through ligand-induced motions that enable the FMN-binding domain to overcome a kinetically unproductive conformation. This is remarkable since the FMN-binding domain is not directly involved in coenzyme binding, the NADP(H) binding site being fully contained in the FAD-binding domain. Sequential rapid mixing measurements indicate that harnessing of coenzyme binding energy to the formation of a kinetically productive CPR-cytochrome c complex is a highly synchronized event. The inferred half-time for the decay of this productive conformation (tau(50)) is 330 +/- 70 ms only. Previously proposed structural and kinetic models are discussed in light of these findings.     

Production of a recombinant hybrid hemoflavoprotein: engineering a functional NADH:cytochrome c reductase.

A gene has been constructed coding for a unique fusion protein, NADH:cytochrome c reductase, that comprises the soluble heme-containing domain of rat hepatic cytochrome b(5) as the amino-terminal portion of the protein and the soluble flavin-containing domain of rat hepatic cytochrome b(5) reductase as the carboxyl terminus. The gene has been expressed in Escherichia coli resulting in the highly efficient production of a functional hybrid hemoflavoprotein which has been purified to homogeneity by a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP agarose, and size-exclusion chromatography. The purified protein exhibited a molecular mass of approximately 46 kDa by polyacrylamide gel electrophoresis and 40,875 Da, for the apoprotein, using mass spectrometry which also confirmed the presence of both heme and FAD prosthetic groups. The fusion protein showed immunological cross-reactivity with both anti-rat cytochrome b(5) and anti-rat cytochrome b(5) reductase antibodies indicating the conservation of antigenic determinants from both native domains. Spectroscopic analysis indicated the fusion protein contained both a b-type cytochrome and flavin chromophors with properties identical to those of the native proteins. Amino-terminal and internal amino acid sequencing confirmed the identity of peptides derived from both the heme- and flavin-binding domains with sequences identical to the deduced amino acid sequence. The isolated fusion protein retained NADH:ferricyanide reductase activity (k(cat) = 8.00 x 10(2) s(-1), K(NADH)(m) = 4 microM, K(FeCN(6))(m) = 11 microM) comparable to that of that of native NADH:cytochrome b(5) reductase and also exhibited both NADH:cytochrome c reductase activity (k(cat) = 2.17 x 10(2) s(-1), K(NADH)(m) = 2 microM, K(FeCN(6))(m) = 11 microM, K(Cyt.c)(m) = 1 microM) and NADH:methemoglobin reductase activity (k(cat) = 4.40 x 10(-1) s(-1), K(NADH)(m) = 3 microM, K(mHb)(m) = 47 microM), the latter two activities indicating efficient electron transfer from FAD to heme and retention of physiological function. This work represents the first successful bacterial expression of a soluble, catalytically competent, rat hepatic cytochrome b(5)-cytochrome b(5) reductase fusion protein that retains the functional properties characteristic of the individual heme and flavin domain.     

Sulfite oxidase from Merluccius productus

Sulfite oxidase (sulfite:oxygen oxidoreductase, EC 1.8.3.1) was purified 482-fold from liver of the Pacific hake Merluccius productus. The molecular weight of the enzyme was found to be 120 000 by gel exclusion chromatography on Sephadex G-100. Electrophoretic analysis on sodium dodecyl sulfate (SDS)-polyacrylamide gel revealed that the enzyme was composed of two subunits whose molecular weight was estimated to be 60 000. The pH optimum of the enzyme was 8.7; Ks for sulfite, 2.5 x 10(-5) M; and that for cytochrome c, 3.6 x 10(-7) M. The enzyme elicited an EPR signal at g = 1.97 characteristic of pentavalent molybdenum. Colorimetric analysis also disclosed that the enzyme contained 2 mol each of heme and molybdenum per mol of protein. This fish liver homogenate in isotonic sucrose solution was fractionated by differential centrifugation into nuclei, mitochondria, microsomes and supernatant (100 000 X g). The major portion of sulfite oxidase activity was found in mitochondria. The sulfite oxidase activity was markedly high in liver and kidney, as compared with that in heart, spleen, muscle, gill and eye.     

Sulfite oxidation catalyzed by aa(3)-type cytochrome c oxidase in Acidithiobacillus ferrooxidans

Sulfite is produced as a toxic intermediate during Acidithiobacillus ferrooxidans sulfur oxidation. A. ferrooxidans D3-2, which posseses the highest copper bioleaching activity, is more resistant to sulfite than other A. ferrooxidans strains, including ATCC 23270. When sulfite oxidase was purified homogeneously from strain D3-2, the oxidized and reduced forms of the purified sulfite oxidase absorption spectra corresponded to those of A. ferrooxidans aa(3)-type cytochrome c oxidase. The confirmed molecular weights of the α-subunit (52.5 kDa), the β-subunit (25 kDa), and the γ-subunit (20 kDa) of the purified sulfite oxidase and the N-terminal amino acid sequences of the γ-subunit of sulfite oxidase (AAKKG) corresponded to those of A. ferrooxidans ATCC 23270 cytochrome c oxidase. The sulfite oxidase activities of the iron- and sulfur-grown A. ferrooxidans D3-2 were much higher than those cytochrome c oxidases purified from A. ferrooxidans strains ATCC 23270, MON-1 and AP19-3. The activities of sulfite oxidase purified from iron- and sulfur-grown strain D3-2 were completely inhibited by an antibody raised against a purified A. ferrooxidans MON-1 aa(3)-type cytochrome c oxidase. This is the first report to indicate that aa(3)-type cytochrome c oxidase catalyzed sulfite oxidation in A. ferrooxidans.     

Correlation of the kinetics of electron transfer activity of various eukaryotic cytochromes c with binding to mitochondrial cytochrome c oxidase.

1. A detailed study of cytochrome c oxidase activity with Keilin-Hartree particles and purified beef heart enzyme, at low ionic strength and low cytochrome c concentrations, showed biphasic kinetics with apparent Km1 = 5 x 10(-8) M, and apparent Km2 = 0.35 to 1.0 x 10(-6) M. Direct binding studies with purified oxidase, phospholipid-containing as well as phospholiptaining aid-depleted, demonstrated two sites of interaction of cytochrome c with the enzyme, with KD1 less than or equal to 10(-7) M, and KD2 = 10(-6) M. 2. The maximal velocities as low ionic strength increased with pH and were highest above ph 7.5. 3. The presence and properties of the low apparent Km phase of the kinetics were strongly dependent on the nature and concentration of the anions in the medium. The multivalent anions, phosphate, ADP, and ATP, greatly decreased the proportion of this phase and similarly decreased the amount of high affinity cytochrome c-cytochrome oxidase complex formed. The order of effectiveness was ATP greater than ADP greater than P1 and since phosphate binds to cytochrome c more strongly than the nucleotides, it is concluded that the inhibition resulted from anion interaction with the oxidase. 4mat low concentrations bakers' yeast iso-1, bakers' yeast iso-1, horse, and Euglena cytochromes c at high concentrations all attained the same maximal velocity. The different proportions of low apparent Km phase in the kinetic patterns of these cytochromes c correlated with the amounts of high affinity complex formed with purified cytochrome c oxidase. 5. The apparent Km for cytochrome c activity in the succinate-cytochrome c reductase system of Keilin-Hartree particles was identical with that obtained with the oxidase (5 x 10(-8) M), suggesting the same site serves both reactions. 6. It is concluded that the observed kinetics result from two catalytically active sites on the cytochrome c oxidase protein of different affinities for cytochrome c. The high affinity binding of cytochrome c to the mitochondrial membrane is provided by the oxidase and at this site cytochrome c can be reduced by cytochrome c1. Physiological concentrations of ATP decrease the affinity of this binding to the point that interaction of cytochrome c with numerous mitochondrial pholpholipid sites can competitively remove cytochrome c from the oxidase. It is suggested that this effect of ATP represents a possible mechanism for the control of electron flow to the oxidase.     

Sulfide dehydrogenase activity of the monomeric flavoprotein SoxF of Paracoccus pantotrophus

Flavocytochrome c-sulfide dehydrogenases (FCSDs) are complexes of a flavoprotein with a c-type cytochrome performing hydrogen sulfide-dependent cytochrome c reduction in vitro. The amino acid sequence analysis revealed that the phylogenetic relationship of different flavoproteins reflected the relationship of sulfur-oxidizing bacteria. The flavoprotein SoxF of Paracoccus pantotrophus is 29-67% identical to the flavoprotein subunit of FCSD of phototrophic sulfur-oxidizing bacteria. Purification of SoxF yielded a homogeneous emerald-green monomeric protein of 42 797 Da. SoxF catalyzed sulfide-dependent horse heart cytochrome c reduction at the optimum pH of 6.0 with a k(cat) of 3.9 s(-1), a K(m) of 2.3 microM for sulfide, and a K(m) of 116 microM for cytochrome c, as determined by nonlinear regression analysis. The yield of 1.9 mol of cytochrome c reduced per mole of sulfide suggests sulfur or polysulfide as the product. Sulfide dehydrogenase activity of SoxF was inhibited by sulfur (K(i) = 1.3 microM) and inactivated by sulfite. Cyanide (1 mM) inhibited SoxF activity at pH 6.0 by 25% and at pH 8.0 by 92%. Redox titrations in the infrared spectral range from 1800 to 1200 cm(-1) and in the visible spectral range from 400 to 700 nm both yielded a midpoint potential for SoxF of -555 +/- 10 mV versus Ag/AgCl at pH 7.5 and -440 +/- 20 mV versus Ag/AgCl at pH 6.0 (-232 mV versus SHE') and a transfer of 1.9 electrons. Electrochemically induced FTIR difference spectra of SoxF as compared to those of free flavin in solution suggested a strong cofactor interaction with the apoprotein. Furthermore, an activation/variation of SoxF during the redox cycles is observed. This is the first report of a monomeric flavoprotein with sulfide dehydrogenase activity.     

顺铂与小鼠艾氏腹水肝癌细胞膜的相互作用

本文研究了顺铂对小鼠艾氏腹水肝癌细胞膜蛋白内源性荧光的淬灭作用和测定了其在膜上的结合量。结果表明顺铂能与癌细胞膜结合。按存在两类结合部位,得到表观结合常数和结合部位数为: K_1=1.35×10~5L/mol n_1=6.80×10~(-4)mol/g(protein) K_2=2.50×10~3L/mol n_2=1.92×10~(-3)mol/g(protein)     

Nitrobacter winogradskyi cytochrome a1c1 is an iron-sulfur molybdoenzyme having hemes a and c

Cytochrome a1c1 (nitrite-cytochrome c oxidoreductase) purified from Nitrobacter winogradskyi (formerly N. agilis) contained molybdenum, non-heme iron, and acid-labile sulfur in addition to hemes a and c; it contained 1 mol of heme a, 4-5 g atoms of non-heme iron, 2-5 g atoms of acid-labile sulfur, and 1-2 g atoms of molybdenum per mol of heme c, but did not contain copper. The fluorescence spectra of the molybdenum cofactor derivative prepared from cytochrome a1c1 were very similar to those of the cofactor derivative from xanthine oxidase, and the aponitrate reductase of nit-1 mutant of Neurospora crassa was complemented by addition of the molybdenum cofactor derived from the cytochrome. Further, the ESR spectrum of cytochrome a1c1 was similar to that of liver sulfite oxidase. The content of cytochrome a1 in the cells cultivated with the medium in which tungsten was substituted for molybdenum markedly decreased as compared with that in the cells cultivated in the molybdenum-supplemented medium. These results indicate that cytochrome a1c1 is an iron-sulfur molybdoenzyme which contains hemes a and c.     

A copper metallochaperone for photosynthesis and respiration reveals metal-specific targets, interaction with an importer, and alternative sites for copper acquisition.

A bacterial two-hybrid assay revealed interaction between a protein now designated bacterial Atx1 and amino-terminal domains of copper-transporting ATPases CtaA (cellular import) and PacS (thylakoid import) but not the related zinc (ZiaA) or cobalt (CoaT) transporters from the same organism (Synechocystis PCC 6803). The specificity of metallochaperone interactions coincides with metal specificity. After reconstitution in a N(2) atmosphere, bacterial Atx1 bound 1 mol of copper mol(-1), and apoPacS(N) acquired copper from copper-Atx1. Copper was displaced from Atx1 by p-(hydroxymercuri)phenylsulfonate, indicative of thiol ligands, and two cysteine residues were obligatory for two-hybrid interaction with PacS(N). This organism contains compartments (thylakoids) where the copper proteins plastocyanin and cytochrome oxidase reside. In copper super-supplemented mutants, photooxidation of cytochrome c(6) was greater in Deltaatx1DeltactaA than in DeltactaA, showing that Atx1 contributes to efficient switching from iron in cytochrome c(6) to copper in plastocyanin for photosynthetic electron transport. Cytochrome oxidase activity was also less in membranes purified from low [copper]-grown Deltaatx1 or DeltapacS, compared with wild-type, but the double mutant Deltaatx1DeltapacS was non-additive, consistent with Atx1 acting via PacS. Conversely, activity in Deltaatx1DeltactaA was less than in either respective single mutant, revealing that Atx1 can function without the major copper importer and consistent with a role in recycling endogenous copper.     

Polypeptide halomethyl ketones bind to serine proteases as analogs of the tetrahedral intermediate. X-ray crystallographic comparison of lysine- and phenylalanine-polypeptide chloromethyl ketone-inhibited subtilisin.

1. A detailed study of cytochrome C oxidse activity with Keilin-Hartree particles and purified beef heart enzyme, at low ionic strength and low cytochrome C concentrations, showed biphasic kinetics with apparent Km1 = 5 x 10(-8) M, and apparent Km2 = 0.35 to 1.0 x 10(-6) M. Direct binding studies with purified oxidase, phospholipid-containing as well as phospholipid-depleted, demonstrated two sites of interaction of cytochrome c with the enzyme, with KD2 less than or equal to 10(-7) M, and KD2 = 10(-6) M. 2...     

Kalisomes in corals: a novel KCl concentrating organelle?

Large membrane-bound inclusions were clearly visible within the gastrodermis and lipid-containing cells of planulae and settled larvae of the zooxanthellate coral, Pocillopora damicornis after fixation or freeze-substitution. We suggest that these inclusions may be a novel potassium (kalium) chloride concentrating organelle, for which we propose the name kalisome. The inclusions were more abundant in settled larvae than in planulae and were not present in mature polyps. In planulae of the azooxanthellate coral, Dendrophyllia sp. these inclusions were extremely rare. Quantitative X-ray microanalysis of freeze-substituted preparations showed that the inclusions in P. damicornis settled larvae contained very high, positively correlated, concentrations of K (2.5 mol x kg(-1)) and Cl (2.5 mol x kg(-1)). Lower concentrations of both K (1.2 mol x kg(-1)) and Cl (1.3 mol x kg(-1)) were detected in P. damicornis planulae, yet higher concentrations were measured in Dendrophyllia planulae (K=6.0 mol x kg(-1); Cl=5.1 mol x kg(-1)). No significant (P>0.05) differences in concentration were observed between inclusions in freeze-substituted and freeze-dried sections of planulae. Symbiotic algae (zooxanthellae) in P. damicornis planulae and settled larvae also contained deposits with high levels of K and Cl, but these were not positively correlated and no structures associated with them were retained by fixation. Significant (P<0.05) concentration differences were also observed between deposits in freeze-substituted and freeze-dried sections. However, similar to 'kalisomes,' zooxanthellae deposits were more abundant in settled larvae than planulae and absent in mature polyps. Higher concentrations of K and Cl were also detected in settled larvae (K=0.7 mol x kg(-1); Cl=1.1 mol x kg(-1)) in comparison to planulae (K=0.4 mol x kg(-1); Cl=0.5 mol x kg(-1)).     

The mutation K30D disrupts the only salt bridge at the subunit interface of the homodimeric hemoglobin from Scapharca inaequivalvis and changes the mechanism of cooperativity.

The subunit interface of the homodimeric hemoglobin from Scapharca inaequivalvis, HbI, is stabilized by a network of interactions that involve several hydrogen-bonded structural water molecules, a hydrophobic patch, and a single, symmetrical salt bridge between residues Lys-30 and Asp-89. Upon mutation of Lys-30 to Asp, the interface is destabilized markedly. Sedimentation equilibrium and velocity experiments allowed the estimate of the dimerization constants for the unliganded (K(1,2D) = 8 x 10(4) M(-1)) and for the CO-bound (K(1,2L) = 1 x 10(3) m(-1)) and oxygenated (K(1,2L) = 70 m(-1)) derivatives. For the oxygenated derivative, the destabilization of the subunit interface with respect to native HbI corresponds to about 8 kcal/mol, an unexpectedly high figure. In the K30D mutant, at variance with the native protein, oxygen affinity and cooperativity are strongly dependent on protein concentration. At low protein concentrations (e.g. 1.2 x 10(-5) m heme), at which the monomeric species becomes significant also in the unliganded derivative, oxygen affinity increases and cooperativity decreases. At protein concentrations where both derivatives are dimeric (e.g. 3.3 x 10(-3) m heme), both cooperativity and oxygen affinity decrease. Taken together, the experimental data indicate that in the K30D mutant, the mechanism of cooperativity is drastically altered and is driven by a ligand-linked monomer-dimer equilibrium rather than being based on a direct heme-heme communication as in native HbI.     

Thermodynamic characterization of the binding of nucleotides to glycyl-tRNA synthetase

The interaction of adenine nucleotides with glycyl-tRNA synthetase was examined by several experimental approaches. ATP and nonsubstrate ATP analogues render glycyl-tRNA synthetase more resistant to digestion by a number of proteases (thrombin, Arg-C, and chymotrypsin) at concentrations that correlate with their Michaelis constants or inhibition constants, consistent with their exerting an effect by binding at the ATP site. Glycine had little effect alone but potentiated the effect of ATP in increasing the resistance to thrombin digestion, consistent with the formation of an enzyme-bound adenylate. No protection from thrombin digestion was afforded by tRNA(gly). Binding constants were determined by isothermal titration calorimetry at 25 degrees C for ATP (2.5 x 10(5) M(-1)), AMPPNP (3.7 x 10(5) M(-1)), and AMPPCP (2.2 x 10(6) M(-1)). The nucleotides had similar values for DeltaH (-71 kJ mol(-1)), with values for TDeltaS that accounted for the differences in the binding constants. Near-ultraviolet CD spectra of the enzyme-nucleotide complexes indicate that the nucleotides are bound in the anti configuration. A glycyl-adenylate analogue, glycine sulfamoyl adenosine (GSAd), bound with a large value for DeltaH (-187 kJ mol(-1)), which was balanced by a large TDeltaS term to give a binding constant (3.7 x 10(6) M(-1)) only slightly larger than that of AMPPCP. Glycine binding to the enzyme could not be detected calorimetrically, and its presence did not change the thermodynamic parameters for binding of AMPPCP. AMPPNP and AMPPCP were not substrates for glycyl-tRNA synthetase. Analysis of the temperature dependence of ATP binding indicated that the heat capacity change is small, whereas the binding of GSAd is accompanied by a large negative heat capacity change (-2.6 kJ K(-1) mol(-1)). Titrations performed in buffers with different ionization enthalpies indicate that the large value for DeltaH for the adenylate analogue does not arise from a coupled protonation event. Differential scanning calorimetry indicated that glycyl-tRNA synthetase is stabilized by nucleotides. Unfolding of the protein is irreversible, and thermodynamic parameters for unfolding could therefore not be determined. The results are consistent with a significant conformational transition in glycyl-tRNA synthetase coupled to the binding of GSAd.     

The redox couple of the cytochrome c cyanide complex: the contribution of heme iron ligation to the structural stability, chemical reactivity, and physiological behavior of horse cytochrome c

Contrary to most heme proteins, ferrous cytochrome c does not bind ligands such as cyanide and CO. In order to quantify this observation, the redox potential of the ferric/ferrous cytochrome c-cyanide redox couple was determined for the first time by cyclic voltammetry. Its E0' was -240 mV versus SHE, equivalent to -23.2 kJ/mol. The entropy of reaction for the reduction of the cyanide complex was also determined. From a thermodynamic cycle that included this new value for the cyt c cyanide complex E0', the binding constant of cyanide to the reduced protein was estimated to be 4.7 x 10(-3) L M(-1) or 13.4 kJ/mol (3.2 kcal/mol), which is 48.1 kJ/mol (11.5 kcal/mol) less favorable than the binding of cyanide to ferricytochrome c. For coordination of cyanide to ferrocytochrome c, the entropy change was earlier experimentally evaluated as 92.4 J mol(-1) K(-1) (22.1 e.u.) at 25 K, and the enthalpy change for the same net reaction was calculated to be 41.0 kJ/mol (9.8 kcal/mol). By taking these results into account, it was discovered that the major obstacle to cyanide coordination to ferrocytochrome c is enthalpic, due to the greater compactness of the reduced molecule or, alternatively, to a lower rate of conformational fluctuation caused by solvation, electrostatic, and structural factors. The biophysical consequences of the large difference in the stabilities of the closed crevice structures are discussed.     

Spectral, kinetic, and thermodynamic properties of Cu(I) and Cu(II) binding by methanobactin from Methylosinus trichosporium OB3b

To examine the potential role of methanobactin (mb) as the extracellular component of a copper acquisition system in Methylosinus trichosporium OB3b, the metal binding properties of mb were examined. Spectral (UV-visible, fluorescence, and circular dichroism), kinetic, and thermodynamic data suggested copper coordination changes at different Cu(II):mb ratios. Mb appeared to initially bind Cu(II) as a homodimer with a comparatively high copper affinity at Cu(II):mb ratios below 0.2, with a binding constant (K) greater than that of EDTA (log K = 18.8) and an approximate DeltaG degrees of -47 kcal/mol. At Cu(II):mb ratios between 0.2 and 0.45, the K dropped to (2.6 +/- 0.46) x 10(8) with a DeltaG degrees of -11.46 kcal/mol followed by another K of (1.40 +/- 0.21) x 10(6) and a DeltaG degrees of -8.38 kcal/mol at Cu(II):mb ratios of 0.45-0.85. The kinetic and spectral changes also suggested Cu(II) was initially coordinated to the 4-thiocarbonyl-5-hydroxy imidazolate (THI) and possibly Tyr, followed by reduction to Cu(I), and then coordination of Cu(I) to 4-hydroxy-5-thiocarbonyl imidazolate (HTI) resulting in the final coordination of Cu(I) by THI and HTI. The rate constant (k(obsI)) of binding of Cu(II) to THI exceeded that of the stopped flow apparatus that was used, i.e., >640 s(-)(1), whereas the coordination of copper to HTI showed a 6-8 ms lag time followed by a k(obsII) of 121 +/- 9 s(-)(1). Mb also solubilized and bound Cu(I) with a k(obsI) to THI of >640 s(-)(1), but with a slower rate constant to HTI (k(obsII) = 8.27 +/- 0.16 s(-)(1)), and appeared to initially bind Cu(I) as a monomer.     

A simple chemical example of hierarchical thermodynamic interactions: the protonation equilibria of inorganic polyprotic acids

A general method for formulating complex thermodynamic systems in terms of hierarchical interactions has been developed, and has been applied in a previous analysis to hemoglobin oxygen binding data. Polyprotic acids can be considered a simple chemical model of thermodynamic interaction between ligand binding events. To further illustrate the hierarchical interaction approach it is applied to the analysis of the thermodynamic interactions between proton binding events in inorganic polyprotic acids. pK values for arsenate, carbonate, chromate, phosphate, phosphite, selenite, sulfide and sulfite were recast into hierarchical interaction terms. The intrinsic K(d,h) for protonation ranged from 8.8 x 10(-13) (M) for phosphate to 1.3 x 10(-6) (M) for chromate. Pairwise interactions (K(d,hh)) between protonation events ranged from 1.3 x 10(4) for phosphite to 9.4 x 10(5) for carbonate. Third order interactions (K(d,hhh)) were 0.91 and 0.51 for arsenate and phosphate, respectively, values relatively close to the no interaction value of 1. A principle feature of systems described by hierarchical interactions is that higher order interactions, representing more complex interactions, are less likely to be significant than lower order interactions, and this is further illustrated by these observations from polyprotic acids. The set of significant hierarchical interaction values can be used to predict values for as yet unobserved events, and projected pK values are made for all the polyprotic acids included in this study. Finally, application of this method to the protonation equilibria of water demonstrates a profound pairwise interaction between protonation events (K(d,hh) = 1.3 x 10(17)), which is attributed to oxygen's small size and lack of polarizability.     

Characterization of electron transfer and proton translocation activities in trypsin-treated bovine heart mitochondrial cytochrome c oxidase

Bovine heart mitochondrial cytochrome c oxidase has been treated with trypsin in order to investigate the role of components a, b, and c (nomenclature of Capaldi) in cytochrome c binding, electron transfer, and proton-pumping activities. Cytochrome c oxidase was dispersed in nondenaturing detergent solution (B. Ludwig, N. W. Downer, and R. A. Capaldi (1979) Biochemistry 18, 1401) and treated with trypsin. This treatment inhibited electron transfer activity by 9% when compared to a similarly treated control in a polarographic assay (493 s-1) and had no large effect on the high affinity (Km = 6.1 X 10(-8) M) or low affinity (Km = 2.2 X 10(-6) M) sites of cytochrome c interaction with cytochrome c oxidase. Direct thermodynamic binding experiments with cytochrome c showed that neither the high affinity (1.04 +/- 0.06 mol cytochrome c/mol cytochrome c oxidase) nor the high-plus-low affinity (2.21 +/- 0.15 mol cytochrome c/mol cytochrome c oxidase) binding sites of cytochrome c on the enzyme were perturbed by the trypsin treatment. Control and trypsin-treated enzyme incorporated into phospholipid vesicles (prepared by the cholate dialysis method) exhibited respiratory control ratios of 6.5 +/- 0.7 and 6.3 +/- 0.6, respectively. The vectorial proton translocation activity in the phospholipid vesicles was unaffected by trypsin treatment with proton translocated to electron transferred ratios being equivalent to the control. NaDodSO4-PAGE showed that components a, b, and c were completely removed by the trypsin treatment. [14C]Iodoacetamide labeling experiments showed that the content of component c in the enzyme was depleted by 85% and that greater than 50% of component a was cleaved upon the trypsin treatment. These results suggest that components a, b, and c are not required for maximum electron transfer and proton translocation activities in the isolated enzyme.