Reduction of methemerythrin-anion adducts by dithionite ion

The reduction by dithionite ion (in excess) of methemerythrin-anion adducts, Hr+X-, to deoxyhemerythrin, Hr degree, has been examined at 25 degrees and pH 6.3 and 8.2. The results accord with the scheme: S2O42- in equilibrium 2SO2- rapid Hr+X- in equilibrium Hr++X- k-1, k1 Hr++SO2- leads to PRODUCT k2 with X- = Br-, HCO2-, CNO-, and F-, k2[SO2-] greater than k1[X-], and the pseudo first-order rate constant, kobs (= k-1), is independent of [X-] and [S2O42-]. Only with X- = NCS- is k2[SO2-] approximately k1[X-] and kobs = a[S2O42-]1/2 (b[NCS-] + [S2OR2-]1/2)-1. Values at pH 6.3 of k-1 (sec-1) and k1 (M-1 sec-1), obtained by anation and anion displacement reactions, are 2.3 x 10(-3), 1.6 x 10(-2) (Br-); 1.5 x 10(-3), 1.2 x 10(-2) (HCO2-); 1.3 x 10(-4), 0.52 (CNO-) and approximately 2 x 10(-4), 3.3 x 10(-3) (CN-, pH 7.0). Values of k-1 from reduction and displacement methods are in good agreement with each other. The value of k2 (1.6 x 10(5) M-1 sec-1, pH 6.3) in somewhat smaller than that for reduction of the met form of hemoproteins. There is only a small effect of pH on rates. Direct reduction of Hr+CN- does not occur, in contrast with Mb+CN-.     

Structural correlation of catecholase-like activities of oxy-bridged dinuclear copper(II) complexes

Eight oxy-bridged dinuclear copper(II) complexes with catecholase-like sites, [Cu(L1)X]2 (HL1 = 1-diethylaminopropan-2-ol, X=N3- 1, NCO- 2, and NO2- 3), [Cu(L2)X]2 (HL2=N-ethylsalicylaldimine, X=NO3- 4, Cl- 5, N3- 6, NCS- 7), and [Cu(L3)]2(ClO4)2, 8 (HL3=N-(salicylidene)-N'-(2-pyridylaldene)propanediamine) have been prepared and characterized. The single crystal X-ray analysis show that the structures of complexes 6 and 8 are dimeric with two adjacent copper(II) atoms bridged by pairs of micro-oxy atoms from the L2 and L3 ligands. Magnetic susceptibility measurements in the temperature range 4-300 K indicate significant antiferromagnetic coupling for 4, 5 and 7 and ferromagnetic coupling for 6 between the copper(II) atoms. The catecholase activity of complexes for the oxidation of 3,5-di-tert-butylcatechol by O2 was studied and it was found that the complexes with the bond distance of Cu(II)...Cu(II) located at 2.9-3.0 A show higher catecholase activity.     

1H NMR spectroscopic characterization of binary and ternary complexes of cobalt(II) carboxypeptidase A with inhibitors

The binding of L- and D-phenylalanine and carboxylate inhibitors to cobalt(II)-substituted carboxypeptidase A, Co(II)CPD (E), in the presence and absence of pseudohalogens (X = N3-, NCO-, and NCS-) has been studied by 1H NMR spectroscopy. This technique monitors the proton signals of histidine residues bound to cobalt(II) and is therefore sensitive to the interactions of inhibitors that perturb the coordination sphere of the metal. Enzyme-inhibitor complexes, E.I, E.I2, and E.I.X, each with characteristic NMR features, have been identified. Thus, for example, L-Phe binds close to the metal ion to form a 1:1 complex, whereas D-Phe binds stepwise, first to a nonmetal site and then to the metal ion to form a 2:1 complex. Both acetate and phenylacetate also form 2:1 adducts stepwise with the enzyme, but beta-phenylpropionate gives a 2:1 complex without any detectable 1:1 intermediate. N3-, NCO-, and NCS- generate E.I.X ternary complexes directly with Co(II)CPD.L-Phe and indirectly with the D-Phe and carboxylate inhibitor 2:1 complexes by displacing the second moiety from its metal binding site. The NMR data suggest that when the carboxylate group of a substrate or inhibitor binds at the active site, a conformational change occurs that allows a second ligand molecule to bind to the metal ion, altering its coordination sphere and thereby attenuating the bidentate behavior of Glu-72. The 1H NMR signals also reflect alterations in the histidine interactions with the metal upon inhibitor binding. Isotropic shifts in the signals for the C-4 (c) and N protons (a) of one of the histidine ligands are readily observed in all of these complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isotope effect evidence for the zinc hydroxide mechanism of carbonic anhydrase catalysis

The carbon kinetic isotope effect on the enzymatic dehydration of HCO3- ion is k12/k13 = 1.011 and is independent, within experimental error, of the addition of sucrose, substitution of D2O for H2O, and substitution of enzyme-bound Zn2+ by Co2+. These results are consistent with a ping-pong mechanism in which proton transfer between enzyme and solvent is separated from HCO3- dehydration. For the dehydration half-reaction, diffusional processes are severalfold faster than dehydration, and the rate-determining step is the dehydration itself. The intrinsic isotope effect is approximately 1.011, indicating that hydration of CO2 occurs by reaction of zinc-bound OH-, rather than zinc-bound H2O.     

Dynamic 13C NMR investigations of substrate interaction and catalysis by cobalt(II) human carbonic anhydrase I

Using 13C NMR spectroscopy, we have further investigated the binding of HCO3- in the active site of an artificial form of human carbonic anhydrase I in which the native zinc is replaced by Co(II). The Co(II) enzyme, unlike all other metal-substituted derivatives, has functional properties closely similar to those of the native zinc enzyme. By measuring the spin-lattice relaxation rate and the line width for both the CO2 and HCO3- at two field strengths, we have determined both the paramagnetic effects that reflect substrate binding and the exchange effects due to catalysis at chemical equilibrium. The following are the results at 14 degrees C and pH 6.3 (1) HCO3- is bound in the active site of the catalytically competent enzyme with the 13C of the HCO3- located 3.22 +/- 0.02 A from the Co(II); (2) the apparent equilibrium dissociation constant for the bound HCO3- is 7.6 +/- 1.5 mM, determined by using the paramagnetic effects on the line widths, and 10 +/- 2 mM, determined by using the exchange effects; (3) the lifetime of HCO3- bound to the metal is (4.4 +/- 0.4) X 10(-5) s; (4) the overall catalyzed CO2 in equilibrium HCO3- exchange rate constant of the Co(II) enzyme is (9.6 +/- 0.4) X 10(3) s-1; (5) the electron spin relaxation time of the Co(II), determined by using paramagnetic effects on the bound HCO3-, is (1.1 +/- 0.1) X 10(-11) s. The data did not provide any direct information on the binding of CO2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystallographic studies of metal ion-DNA interactions: different binding modes of cobalt(II), copper(II) and barium(II) to N7 of guanines in Z-DNA and a drug-DNA complex.

Metal ion coordination to nucleic acids is not only required for charge neutralization, it is also essential for the biological function of nucleic acids. The structural impact of different metal ion coordinations of DNA helices is an open question. We carried out X-ray diffraction analyses of the interactions of the two transition metal ions Co(II) and Cu(II) and an alkaline earth metal ion Ba(II), with DNA of different conformations. In crystals, Co(II) ion binds exclusively at the N7 position of guanine bases by direct coordination. The coordination geometry around Co(II) is octahedral, although some sites have an incomplete hydration shell. The averaged Co-N7 bond distance is 2.3 A. The averaged Co-N7-C8 angle is 121 degrees, significantly smaller than the value of 128 degrees if the Co-N7 vector were to bisect the C5-N7-C8 bond angle. Model building of Co(II) binding to guanine N7 in B-DNA indicates that the coordinated waters in the axial positions would have a van der Waals clash with the neighboring base on the 5' side. In contrast, the major groove of A-DNA does not have enough room to accommodate the entire hydration shell. This suggests that Co(II) binding to either B-DNA or A-DNA may induce significant conformational changes. The Z-DNA structure of Cu(II)-soaked CGCGTG crystal revealed that the Cu(II) ion is bis-coordinated to N7 position of G10 and #G12 (# denotes a symmetry-related position) bases with a trigonal bipyramid geometry, suggesting a possible N7-Cu-N7 crosslinking mechanism. A similar bis-coordination to two guanines has also been seen in the interaction of Cu(II) in m5CGUAm5CG Z-DNA crystal and of Ba(II) with two other Z-DNA crystals.     

Paramagnetic carbon-13 NMR relaxation studies on the kinetics and mechanism of the HCO3-/CO2 exchange catalyzed by manganese(II) human carbonic anhydrase I

A detailed analysis of the stability and activity of Mn(II) human carbonic anhydrase I and the kinetics and mechanism of its catalysis of the HCO3-/CO2 exchange have been performed at pH 8.5. The analysis was based on the paramagnetic relaxation rates R1p and R2p of the 13C atom of HCO3- in the Mn2+/apoenzyme/HCO3-/CO2 system and the HCO3(-)----CO2 interconversion rate obtained by the magnetization-transfer technique. The R1p and R2p rates were measured as functions of the temperature, magnetic field strength, and substrate and apoenzyme concentrations and were interpreted on the basis of the Solomon-Bloembergen-Morgan theories and general equations for the ligand exchange [Led, J. J., & Grant, D. M. (1977) J. Am. Chem. Soc. 99, 5845-5858]. From the analysis of the data, a formation constant for the Mn(II) enzyme of log KMAM = 5.8 +/- 0.4 was obtained while the activity of the Mn(II) enzyme, measured as the HCO3-/CO2 interconversion rate at [HCO3-] = 0.100 M and pH 8.5, was found to be about 4% of that of the native Zn(II) enzyme. However, an effective dissociation constant KeffHCO3- less than or approximately 12 mM and a maximal exchange rate constant kcatexch approximately equal to 400 s-1, also derived by the analysis, result in an apparent second-order rate constant kcatexch/KeffHCO3- only a factor of 4 smaller than the corresponding rate constant for the native Zn(II) isoenzyme I. Most conspicuously, the resulting distance of only 2.71 +/- 0.03 A between the Mn2+ ion of the enzyme and the 13C atom of HCO3- in the enzyme-bicarbonate complex indicates that the bicarbonate is bound to the metal ion by two of its oxygen atoms in the central catalytic step, thereby supporting the modified Zn(II)-OH mechanism [Lindskog, S., Engberg, P., Forsman, C., Ibrahim, S. A., Jonsson, B.-H., Simonsson, I., & Tibell, L. (1984) Ann. N.Y. Acad. Sci. 429, 61-75 (and references cited therein)]. In contrast, this binding mode differs from the structure of the complexes suggested in the rapid-equilibrium kinetic model [Pocker, Y., & Deits, T. L. (1983) J. Am. Chem. Soc. 105, 980-986; Pocker, Y., & Deits, T. L. (1984) Ann. N.Y. Acad. Sci. 429, 76-83].     

The role of CO2 in cobalt-catalyzed peroxidations

Augmentation, by CO(2)/HCO(3)(-), of Co(II)-catalyzed peroxidations was explored to clarify whether the rate enhancement was due to CO(2) or to HCO(3)(-). The rate of oxidation of NADH by Co(II) plus H(2)O(2), in Tris or phosphate, was markedly enhanced by CO(2)/HCO(3)(-). Phosphate was seen to inhibit the Co(II)-catalyzed peroxidation, probably due to its sequestration of the Co(II). When CO(2) was used, there was an initial burst of NADH oxidation followed by a slower linear rate. The presence of carbonic anhydrase eliminated this initial burst; establishing that CO(2) rather than HCO(3)(-) was the species responsible for the observed rate enhancements. Both kinetic and spectral data indicated that Co(II) was converted by H(2)O(2) into a less active form from which Co(II) could be regenerated. This less active form absorbed in both the UV and visible regions, and is assumed to be a peroxy bridged binuclear complex. The rate of formation of this absorbing form was increased by HCO(3)(-)/CO(2). A minimal mechanism consistent with these observations is proposed.     

Origin of viscosity effects in carbonic anhydrase catalysis. Kinetic studies with bulky buffers at limiting concentrations

In our earlier paper we showed that the rates of CO2 hydration and HCO3- dehydration catalyzed by the high-activity form of mammalian erythrocyte carbonic anhydrase (CA II) were dependent on solution viscosity increase and that the effect was linked to some kind of proton-transfer-related event [Pocker, Y., & Janji?, N. (1987) Biochemistry 26, 2597-2606]. In order to further elucidate the source of the observed viscosity effect, the dependence of kcat and Km for CA II catalyzed HCO3- dehydration at pH 5.90 on sucrose-induced viscosity increase was investigated at several concentrations of 2-(N-morpholino)ethanesulfonic acid (MES) buffer, including the very low buffer concentration region (less than 10 mM) where the proton transfer between the shuttle group on the enzyme and buffer becomes rate limiting. In all examined cases, kcat steadily decreased with added sucrose while Km remained independent of the viscosity increase. The extent to which this reaction was dependent on viscosity was found to be constant, within experimental error, over the entire range of MES buffer concentrations studied (1-20 mM). Furthermore, the viscosity effect was qualitatively and quantitatively the same when an exceptionally large buffer (i.e., bovine serum albumin) was used instead of the more commonly used biological buffer (i.e., MES).(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of a square-planar Co(I) B12 intermediate. Implications for enzyme catalysis.

X-ray edge and extended x-ray absorption fine structure (EXAFS) techniques provide powerful tools for analysis of local molecular structure of complexes in solution. We present EXAFS results for Co(I) B12 that demonstrate a four-coordinate (distorted) square-planar configuration. Comparison of EXAFS solutions for Co(I) and Co(II) B12 (collected previously; Sagi et al. 1990. J. Am. Chem. Soc. 112:8639-8644) suggest that modulation of the Co-N bond to the axial 5,6-dimethylbenzimidazole (DMB), in the absence of changes in Co-N (equatorial) bond distances, may be a key mechanism in promoting homolytic versus heterolytic cleavage. As Co-C bond homolysis occurs, the Co-N (DMB) bond becomes stronger. However, for heterolytic cleavage to occur, earlier electrochemical studies (D. Lexa and J. M. Saveant. 1976. J. Am. Chem. Soc. 98:2652-2658) and recent studies of methylcobalamin-dependent Clostridium thermoaceticum (Ragsdale et al. 1987. J. Biol. Chem. 262:14289-14297) suggest that removal of the DMB ligand (before Co-C bond cleavage) favors formation of the four-coordinate square-planar Co(I) species while inhibiting formation of the five-coordinate Co(II) B12 complex. This paper presents the first direct evidence that formation of the Co(I) B12 intermediate must involve breaking of the Co-N (DMB) bond.     

Catalysis by cobalt(II)-substituted carbonic anhydrase II of the exchange of oxygen-18 between CO2 and H2O

We have measured the catalysis by Co(II)-substituted bovine carbonic anhydrase II from red cells of the exchange of 18O between CO2 and H2O using membrane-inlet mass spectrometry. We chose Co(II)-substituted carbonic anhydrase II because the apparent equilibrium dissociation constant of HCO3- and enzyme at pH 7.4, KHCO3-eff approximately equal to 55 mM, was within a practicable range of substrate concentrations for the 18O method. For the native, zinc-containing enzyme KHCO3-eff is close to 500 mM at this pH. The rate constant for the release from the active site of water bearing substrate oxygen kH2O was dependent on the fraction of enzyme that was free, not bound by substrate HCO3- or anions. The pH dependence of kH2O in the pH range 6.0-9.0 can be explained entirely by a rate-limiting, intramolecular proton transfer between cobalt-bound hydroxide and a nearby group, probably His-64. The rate constant for this proton transfer was found to be 7 X 10(5) S-1 for the Co(II)-substituted enzyme and 2 X 10(6) S-1 for the native enzyme. These results are applied to models derived from proton-relaxation enhancement of water exchanging from the inner coordination shell of the cobalt in carbonic anhydrase. The anions iodide, cyanate, and thiocyanate inhibited catalysis of 18O exchange by Co(II)-substituted carbonic anhydrase II in a manner competitive with total substrate (CO2 and HCO3-) at chemical equilibrium and pH 7.4. These results are discussed in terms of observed steady-state inhibition patterns and suggest that there is no significant contribution of a ternary complex between substrate, inhibitor, and enzyme.     

Mechanistic studies on metal-pyrophosphate hydrolysis. Structure of tetraammine(chloroaquo)cobalt (III) dichloride ([Co(NH3)4ClH2O]2+.2Cl-), an acid hydrolysis product of Co(NH3)4HP2O7 and Co(NH3)4PO4

The octahedral complex tetraammine(chloroaquo)cobalt(III) dichloride is shown to be the HCl hydrolysis product of both P1,2-bidentate tetraammine(pyrophosphato)cobalt(III) [Co(NH3)4HP2O7 or CoPP] and bidentate tetraammine(phosphato)cobalt(III) [Co(NH3)4PO4 or CoP]. The complex crystallizes in the orthorhombic space group Pna21 with cell dimensions a = 13.033(2)A, b = 6.710(1)A, and c = 10.318(2)A; the crystal structure was refined to a final disagreement index of 0.033. The average of the four Co-N distances is 1.944 +/- 6A. The Co-Cl distance is 2.257(2)A and the Co-O(W) distance is 1.971(4)A. Both protons of the coordinated water molecule are engaged in strong hydrogen bonds to the two nonbonded chloride counterions with O(W)-Cl distances of 3.087(6)A and 3.123(6)A. Each nonbonded chloride is engaged in seven hydrogen bonding interactions resulting from the high ratio of hydrogen bond donors to acceptors in the CoP structure. Cobalt bisphosphate (CoP2) is the final enzyme hydrolysis product when CoPP is used as substrate in the yeast inorganic pyrophosphatase reaction. The bridge oxygen atom is the site of initial CoPP cleavage both for HCl catalyzed hydrolysis as well as for enzyme catalyzed hydrolysis.     

Buffer enhancement of proton transfer in catalysis by human carbonic anhydrase III

Among the isozymes of carbonic anhydrase, isozyme III is the least efficient in the catalysis of the hydration of CO2 and was previously thought to be unaffected by proton transfer from buffers to the active site. We report that buffers of small size, especially imidazole, increase the rate of catalysis by human carbonic anhydrase III (HCA III) of (1) 18O exchange between HCO3- and water measured by membrane-inlet mass spectrometry and (2) the dehydration of HCO3- measured by stopped-flow spectrophotometry. Imidazole enhanced the rate of release of 18O-labeled water from the active site of wild-type carbonic anhydrase III and caused a much greater enhancement, up to 20-fold, for the K64H, R67H, and R67N mutants of this isozyme. Imidazole had no effect on the rate of interconversion of CO2 and HCO3- at chemical equilibrium. Steady-state measurements showed that the addition of imidazole resulted in increases in the turnover number (kcat) for the hydration of CO2 catalyzed by HCA III and for the dehydration of HCO3- catalyzed by R67N HCA III. These results are consistent with the transfer of a proton from the imidazolium cation to the zinc-bound hydroxide at the active site, a step required to regenerate the active form of enzyme in the catalytic cycle. Like isozyme II of carbonic anhydrase, isozyme III can be enhanced in catalytic rate by the presence of small molecule buffers in solution.     

In situ characterization of carbonic anhydrase activity in isolated rat lungs

Lung carbonic anhydrase (CA) participates directly in plasma CO2-HCO3(-)-H+ reactions. To characterize pulmonary CA activity in situ, CO2 excretion and capillary pH equilibration were examined in isolated saline-perfused rat lungs. Isolated lungs were perfused at 25, 30, and 37 degrees C with solutions containing various concentrations of HCO3- and a CA inhibitor, acetazolamide (ACTZ). Total CO2 excretion was partitioned into those fractions attributable to dissolved CO2, uncatalyzed HCO3- dehydration, and catalyzed HCO3- dehydration. Approximately 60% of the total CO2 excretion at each temperature was attributable to CA-catalyzed HCO3- dehydration. Inhibition of pulmonary CA diminished CO2 excretion and produced significant postcapillary perfusate pH disequilibria, the magnitude and time course of which were dependent on temperature and the extent of CA inhibition. The half time for pH equilibration increased from approximately 5 s at 37 degrees C to 14 s at 25 degrees C. For the HCO3- dehydration reaction, pulmonary CA in situ displayed an apparent inhibition constant for ACTZ of 0.9-2.2 microM, a Michaelis-Menten constant of 90 mM, a maximal reaction velocity of 9 mM/s, and an apparent activation energy of 3.0 kcal/mol.     

The effects of exogenous extracellular carbonic anhydrase on CO2 excretion in rainbow trout (Oncorhynchus mykiss): role of plasma buffering capacity

The buffering capacity (beta) of rainbow trout (Oncorhynchus mykiss) plasma was manipulated prior to intravascular injection of bovine carbonic anhydrase to test the idea that proton (H+) availability limits the catalysed dehydration of HCO3- within the extracellular compartment. An extracorporeal blood shunt was employed to continuously monitor blood gases in vivo in fish exhibiting normal plasma beta (-3.9+/-0.3 mmol 1(-1) pH unit(-1)), and in fish with experimentally (using N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) elevated plasma beta (-12.1+/-1.1 mmol 1(-1) pH unit(-1)). An injection of 5 mg kg(-1) carbonic anhydrase equally reduced (after 90 min) the arterial partial pressure of CO2 in trout with regular (-0.23+/-0.05 Torr) or high (-0.20+/-0.05 Torr) plasma beta; saline injection was without effect. Because ventilation and venous blood gases were unaffected by carbonic anhydrase, the effect of extracellular carbonic anhydrase in lowering arterial partial pressure of CO2 was likely caused solely by a specific enhancement of CO2 excretion owing to acceleration of HCO3- dehydration within the plasma. The lowering of arterial partial pressure of CO2 in trout after injection of exogenous carbonic anhydrase provides the first in vivo evidence that the accessibility of plasma HCO3- to red blood cell carbonic anhydrase constrains CO2 excretion under resting conditions. Because the velocity of red blood cell Cl-/HCO3- exchange governs HCO3- accessibility to red blood cell carbonic anhydrase, the present study also provides evidence that CO2 excretion at rest is limited by the relatively slow rate of Cl-/HCO3- exchange. The effect of carbonic anhydrase in lowering arterial partial pressure of CO2 was unrelated to plasma buffering capacity. While these data could suggest that H+ availability does not limit extracellular HCO3- dehydration in vivo at resting rates of CO2 excretion, it is more likely that the degree to which plasma beta was elevated in the present study was insufficient to drive a substantially increased component of HCO3- dehydration through the plasma.     

Synthesis, characterization, and antibacterial activity of novel Mn(II), Co(II), Ni(II), Cu(II), and Zn(II) complexes with vitamin K3-thiosemicarbazone

Vitamin K3-thiosemicarbazone (C12H11N3NaO4S2 x 5H2O, abbreviated as VT), a new Schiff base derivative, has been synthesized. Its crystal structure, determined by X-ray diffraction, is triclinic, space group P1. We have also prepared five novel complexes of VT with transition metals: [M(VT)(2)2H2O] x nH2O, (n = 1 and 2 for M = Cu(II) and Zn(II), respectively) and [M'(HVT)2Cl2] x mH2O, (m = 4, 5, and 7 for M' = Co(II), Mn(II), and Ni(II), respectively). These compounds were characterized by IR and UV-Vis spectroscopy, molar conductivity, thermal analyses, complexometric titration, and elemental analysis. In all the complexes, the VT ligand coordinates through sulfur and oxygen atoms, and the geometry around metal atom is best described as octahedral. In vitro tests of antibacterial activity showed that VT and its complexes with Mn(II), Co(II), Ni(II), Cu(II), and Zn(II) all had strong inhibitory actions against G(+) Staphylococcus aureus, G(+) Hay bacillus, and G(-) Escherichia coli.     

Synthesis and crystal structures of Boc-L-Asn-L-Pro-OBzl.CH3OH and dehydration side product, Boc-beta-cyano-L-alanine-L-Pro-OBzl

Boc-L-Asn-L-Pro-OBzl: C21H29O6N3.CH3OH, Mr = 419.48 + CH3 OH, monoclinic, P2(1), a = 10.049(1), b = 10.399(2), c = 11.702(1) A, beta = 92.50(1)degrees, V = 1221.7(3) A3, dx = 1.14 g.cm-3, Z = 2, CuK alpha (lambda = 1.54178 A), F(000) = 484 (with solvent), 23 degrees, unique reflections (I greater than 3 sigma(I)) = 1745, R = 0.043, Rw = 0.062, S = 1.66. Boc-beta-cyano-L-alanine-L-Pro-OBzl: C21H27O5N3, Mr = 401.46, orthorhombic, P2(1)2(1)2(1), a = 15.741(3), b = 21.060(3), c = 6.496(3) A, V = 2153(1) A3, dx = 1.24 g.cm-3, Z = 4, CuK alpha (lambda = 1.54178 A), F(000) = 856, 23 degrees, unique reflections (I greater than 3 sigma(I)) = 1573, R = 0.055, Rw = 0.078, S = 1.86. The tert.-butyloxycarbonyl (Boc) protected dipeptide benzyl ester (OBzl), Boc-L-Asn-L-Pro-OBzl, prepared from a mixed anhydride reaction using isobutylchloroformate, Boc-L-asparagine, and HCl.L-proline-OBzl, crystallized with one methanol per asymmetric unit in an extended conformation with the Asn-Pro peptide bond trans. Intermolecular hydrogen bonding occurs between the methanol and the Asn side chain and between the peptide backbone and the Asn side chain. A minor impurity due to the dehydration of the Asn side chain to a beta-CNala crystallized with a similar extended conformation and a single intermolecular hydrogen bond.     

Bestrophin Cl- channels are highly permeable to HCO3-

Bestrophin-1 (Best1) is a Cl(-) channel that is linked to various retinopathies in both humans and dogs. Dysfunction of the Best1 Cl(-) channel has been proposed to cause retinopathy because of altered Cl(-) transport across the retinal pigment epithelium (RPE). In addition to Cl(-), many Cl(-) channels also transport HCO3(-). Because HCO3(-) is physiologically important in pH regulation and in fluid and ion transport across the RPE, we measured the permeability and conductance of bestrophins to HCO3(-) relative to Cl(-). Four human bestrophin homologs (hBest1, hBest2, hBest3, and hBest4) and mouse Best2 (mBest2) were expressed in HEK cells, and the relative HCO3(-) permeability (P HCO3/PCl) and conductance (G HCO3/GCl) were determined. P HCO3/PCl was calculated from the change in reversal potential (Erev) produced by replacing extracellular Cl(-) with HCO3(-). hBest1 was highly permeable to HCO3(-) (P HCO3)/PCl = approximately 0.44). hBest2, hBest4, and mBest2 had an even higher relative HCO3(-) permeability (P HCO3/PCl = 0.6-0.7). All four bestrophins had HCO3(-) conductances that were nearly the same as Cl(-) (G HCO3/GCl = 0.9-1.1). Extracellular Na+ did not affect the permeation of hBest1 to HCO3(-). At physiological HCO3(-) concentration, HCO3(-) was also highly conductive. The hBest1 disease-causing mutations Y85H, R92C, and W93C abolished both Cl(-) and HCO3(-) currents equally. The V78C mutation changed P HCO3/PCl and G HCO3/GCl of mBest2 channels. These results raise the possibility that disease-causing mutations in hBest1 produce disease by altering HCO3(-) homeostasis as well as Cl(-) transport in the retina.     

Investigation of the system copper(II) carbonic anhydrase and HCO3-/CO2

Copper(II) substituted human and bovine carbonic anhydrases B in the presence of bicarbonate have been investigated in solution through water-solvent proton nuclear magnetic resonance (nmr) at variable magnetic fields. HCO3-, contrary to all the other monoanionic inhibitors, partially reduces the water proton relaxation rates. This has been accounted for on the basis of the availability within the active cavity of two coordination positions partially overlapping. 13C-nmr measurements on both CO2 and HCO3- confirm that HCO3- binds the metal, whereas CO2 interacts with the paramagnetic center at nonbonding distance. The upper limit for the CO2 in equilibrium HCO3- interconversion has been estimated to be 10 sec-1.