Anomalous driving force for renal brush border H+/OH-transport characterized by using 6-carboxyfluorescein

The pH, delta pH, and membrane potential dependences of H+/OH-permeability in renal brush border membrane vesicles (BBMV) were studied by using the entrapped pH indicator 6-carboxyfluorescein (6CF). Quantitative H+/OH-fluxes (JH) were obtained from a calibration of the fluorescence response of 6CF to intravesicular pH using vesicles prepared with varying intravesicular and solution pHs. Intravesicular buffer capacity, determined by titration of lysed vesicles, increased monotonically from 140 to 260 mequiv/L in the pH range 5-8. JH was measured by subjecting voltage-clamped BBMV (K+/valinomycin) to preformed pH gradients over the pH range 5-8 and measuring the rate of change of intravesicular pH. For small preformed pH gradients (0.4 pH unit) JH [6 nequiv s-1 (mg of protein)-1] was nearly independent of pH (5-8), predicting a highly pH dependent H+ permeability coefficient. JH increased in a curvilinear manner from 6 to 104 nequiv s-1 (mg of protein)-1 as delta pH increased from 0.4 to 2.5. JH increased linearly [1.6-7.3 nequiv s-1 (mg of protein)-1] with induced K+ diffusion potentials (21-83 mV) in the absence of a pH gradient. These findings cannot be explained by simple diffusion of H+ or OH- or by mobile carrier models. Two mechanisms are proposed, including a lipid diffusion mechanism, facilitated by binding of H+/OH- to fixed sites in the membrane, and a linear H2O strand model, where dissociation of H2O in the membrane fixes H+ and OH- concentrations in strands, which can result in net H+/OH- transport.     

Purple acid phosphatase from bovine spleen. Interactions at the active site in relation to the reaction mechanism.

Oxidation of the reduced (pink) phosphate-free bovine spleen acid phosphatase with 1.5 mol H2O2 or sodium peroxodisulfate/mol, in the presence of Mes or Bistris pH 5, leads to a species with an absorption maximum at 558 nm. Addition of acetate or oxidation in the presence of acetate buffer engenders a species with a maximum at 550 nm. Addition of phosphate to both species shifts the maximum immediately to 540 nm; this is the species also found after preparation from the spleen. The assumption that these species represent strongly bidentate-binding hydroxo, acetato and phosphato complexes of the Fe(III)-Fe(III) system is supported by replacement reactions with other ligating oxoanions followed by their typical spectral shifts. These oxoanion complexes cannot be dissociated by gel filtration; this is possible only after reduction to the Fe(II)-Fe(III) system. The oxidized species without EPR signals below g values of 2 still reveals 5% activity which cannot be reduced to zero even in the presence of higher concentrations of peroxodisulfate. The pH optimum of the reaction with alpha-naphthyl phosphate shifts from 5.9 to 5.3 in the oxidized species. The apparent pK values around 4.5 as derived from the pH dependence of activity, of the EPR spectra, and the spectral shifts of the phosphate-saturated reduced and oxidized species are assigned to an aquo/hydroxo equilibrium at the Fe(III) or an equilibrium, where the phosphato ligand is replaced by a hydroxo ligand. A reaction mechanism is proposed in which a hydroxo ligand at the chromophoric Fe(III) attacks the phosphoric acid ester group only when that is monoprotonated and pre-oriented by electrostatic interaction with the nonchromophoric metal ion. Binding and inhibition studies with the oxoanions indicate that they compete with the catalytically active hydroxo group of the reduced and oxidized enzyme with nearly the same inhibition constants. Catalysis is not affected by the oxoanions which replace the additional mu-hydroxo ligand in the 558-nm-absorbing Fe(III)-Fe(III) species. In contrast to hemerythrin and ribonucleotide reductase, a binuclear iron center is proposed for the purple acid phosphatase, which is bridged by a carboxylato and two aquo/hydroxo groups, but without a mu-oxo bridge.     

EPR and ENDOR evidence for a 1-His,hydroxo-bridged mixed-valent diiron site in Desulfovibrio vulgaris rubrerythrin

Key features differentiating the coordination environment of the two irons in the mixed-valent (Fe(2+),Fe(3+)) diiron site of Desulfovibrio vulgaris rubrerythrin (Rbr(mv)) were determined by continuous wave (CW) and pulsed ENDOR spectroscopy at 35GHz. (14)N ENDOR evidence indicates that a nitrogen is bound only to the Fe(2+) ion of the mixed-valent site. Assuming that this nitrogen is from His131Ndelta, the same one that furnishes an iron ligand in the crystal structure of the diferric site, the ENDOR data allow us to specify the Fe(2+) and Fe(3+) positions within the molecular reference frame. In addition, the (1,2)H ENDOR on Rbr(mv) indicates the presence of a solvent-derived aqua/hydroxo ligand bound either terminally or in a bridging mode to Fe(3+) in the mixed-valent site. The relatively large g anisotropy of Rbr(mv) and weak antiferromagnetic coupling, J approximately -8 cm(-)(1) (in the 2JS(1)*S(2) formalism), between the irons is more consistent with a bridging than terminal hydroxo ligand. gamma-Irradiation was used to cryoreduce Rbr at 77 K, thereby producing a mixed-valent diiron site [(Rbr(ox))(mv)] that retains the structure of the diferric site. The EPR spectrum of (Rbr(ox))(mv) was nearly identical to that of the as-isolated or chemically reduced samples. This near identity implies that the structure of the mixed-valent Rbr diiron site is essentially identical to that of the diferric site, except for protonation of the oxo bridge, which apparently occurred via a proton jump from hydrogen-bonded solvent at 77 K. The EPR spectrum of (Rbr(ox))(mv) thus supports the (14)N ENDOR-assigned His131 ligation to Fe(2+) and assignment of the solvent-derived ligand observed in the (1,2)H ENDOR to a hydroxo bridge between the irons of the mixed-valent diiron site.     

Electrogenic H+/OH- movement across phospholipid vesicles measured by spin-labeled hydrophobic ions.

Transmembrane pH gradients created across phospholipid vesicles give rise to time-dependent potentials as determined from the EPR spectra of phosphonium ion spin labels in the system. From the time-dependent spectra, the transmembrane H+/OH- current is obtained and hence the current-voltage curve for the vesicle membrane is obtained. The current-voltage curve is linear with a membrane resistance of 3 +/- 2 X 10(9) omega cm2 corresponding to a membrane permeability of 5 +/- 2 X 10(-7) cm/s. This unusually high permeability is further increased by small amounts of lipid oxidation, CHCl3 or the general anesthetic halothane.     

Mechanism of basolateral membrane H+/OH-/HCO-3 transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process

In order to examine the mechanism of basolateral membrane H+/OH-/HCO-3 transport, a method was developed for the measurement of cell pH in the vivo doubly microperfused rat proximal convoluted tubule. A pH-sensitive fluorescein derivative, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein, was loaded into cells and relative changes in fluorescence at two excitation wavelengths were followed. Calibration was accomplished using nigericin with high extracellular potassium concentrations. When luminal and peritubular fluids were pH 7.32, cell pH was 7.14 +/- 0.01. Decreasing peritubular pH from 7.32 to 6.63 caused cell pH to decrease from 7.16 +/- 0.02 to 6.90 +/- 0.03. This effect occurred at an initial rate of 2.4 +/- 0.3 pH units/min, and was inhibited by 0.5 mM SITS. Lowering the peritubular sodium concentration from 147 to 25 meq/liter caused cell pH to decrease from 7.20 +/- 0.03 to 6.99 +/- 0.01. The effect of peritubular sodium concentration on cell pH was inhibited by 0.5 mM SITS, but was unaffected by 1 mM amiloride. In addition, when peritubular pH was decreased in the total absence of luminal and peritubular sodium, the rate of cell acidification was 0.2 +/- 0.1 pH units/min, a greater than 90% decrease from that in the presence of sodium. Cell depolarization achieved by increasing the peritubular potassium concentration caused cell pH to increase, an effect that was blocked by peritubular barium or luminal and peritubular sodium removal. Lowering the peritubular chloride concentration from 128 to 0 meq/liter did not affect cell pH. These results suggest the existence of an electrogenic, sodium-coupled H+/OH-/HCO-3 transport mechanism on the basolateral membrane of the rat proximal convoluted tubule.     

Non-ohmic proton conductance of mitochondria and liposomes

Direct measurements of the proton/hydroxyl ion flux across rat liver mitochondria and liposome membranes are reported. H+/OH- fluxes driven by membrane potential (delta psi) showed nonlinear dependence on delta psi both in mitochondria and in liposomes whereas delta pH-driven H+/OH- flux shows linear dependence on delta pH in liposomes. In the presence of low concentrations of a protonophore the H+/OH- flux was linearly dependent on delta psi and showed complex dependence on delta pH. The nonlinearity of H+/OH- permeability without protonophore is described by an integrated Nernst- Plank equation with trapezoidal energy barrier. Permeability coefficients depended on the driving force but were in the range 10(-3) cm/s for mitochondria and 10(-4)-10(-6) cm/s for liposomes. The nonlinear dependence of H+/OH- flux on delta psi explains the nonlinear dependence of electrochemical proton gradient on the rate of electron transport in energy coupling systems.     

Kinetic isotope effect studies on aspartate aminotransferase: evidence for a concerted 1,3 prototropic shift mechanism for the cytoplasmic isozyme and L-aspartate and dichotomy in mechanism

The C alpha primary hydrogen kinetic isotope effects (C alpha-KIEs) for the reaction of the cytoplasmic isozyme of aspartate aminotransferase (cAATase) with [alpha-2H]-L-aspartate are small and only slightly affected by deuterium oxide solvent (DV = 1.43 +/- 0.03 and DV/KAsp = 1.36 +/- 0.04 in H2O; DV = 1.44 +/- 0.01 and DV/KAsp = 1.61 +/- 0.06 in D2O). The D2O solvent KIEs (SKIEs) are somewhat larger and are essentially independent of deuterium at C alpha (D2OV = 2.21 +/- 0.07 and D2OV/KAsp = 1.70 +/- 0.03 with [alpha-1H]-L-aspartate; D2OV = 2.34 +/- 0.12 and D2OV/KAsp = 1.82 +/- 0.06 with [alpha-2H]-L- aspartate). The C alpha-KIEs on V and on V/KAsp are independent of pH from pH 5.0 to pH 10.0. These results support a rate-determining concerted 1,3 prototropic shift mechanism by the multiple KIE criteria [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106]. The large C alpha-KIEs for the reaction of mitochondrial AATase (mAATase) with L-glutamate (DV = 1.88 +/- 0.13 and DV/KGlu = 3.80 +/- 0.43 in H2O; DV = 1.57 +/- 0.05 and DV/KGlu = 4.21 +/- 0.19 in D2O) coupled with the relatively small SKIEs (D2OV = 1.58 +/- 0.04 and D2OV/KGlu = 1.25 +/- 0.05 with [alpha-1H]-L-glutamate; D2OV = 1.46 +/- 0.06 and D2OV/KGlu = 1.16 +/- 0.05 with [alpha-2H]-L-glutamate) are most consistent with a two-step mechanism for the 1,3 prototropic shift for this isozyme-substrate pair.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of the conserved residues His-246, His-199, and Tyr-255 in the catalysis of catechol 2,3-dioxygenase from Pseudomonas stutzeri OX1

Catechol 2,3-dioxygenase (C2,3O) from Pseudomonas stutzeri OX1, which is able to grow on various aromatic substrates as the sole source of carbon and energy, has been expressed in Escherichia coli, purified, characterized, and found to be very similar to other dioxygenases from Pseudomonas species. Interestingly, the activity of the protein shows a rather unusual pH dependence when assayed on catechol. A model of the catalytic mechanism was developed that is able to reproduce the catalytic behavior of the protein as a function of the pH. The model includes multiple equilibria and four productive intermediates with different ionization states of the enzyme-substrate complex. The fitting of the theoretical curve to the experimental data suggests that a tyrosine and two histidine residues are involved in catalysis. Mutants (H246N)-, (H246A)-, (H199N)- and (Y255F)-C2,3O were produced to investigate the role of highly conserved His-199, His-246, and Tyr-255. The strongly reduced activity of the mutants suggests a primary catalytic role for each of these residues. Moreover, mutants at positions 199 and 246 display pH profiles different from that of the wild-type protein, thus indicating that residues His-246 and His-199 play a role in determining the unusual pH dependence of the enzyme. In addition, electron-withdrawing groups on catechol, which increase the acidity of the phenolic hydroxyl group, are able to counterbalance the effect of the mutation H246N in reducing catalytic activity but cause a further reduction of the activity of (H199N)-C2,3O. This finding suggests that His-246 is involved in the initial catechol deprotonation, whereas His-199 promotes the reaction between oxygen and the aromatic ring.     

Deuterium isotope effects during carbon-hydrogen bond cleavage by trimethylamine dehydrogenase. Implications for mechanism and vibrationally assisted hydrogen tunneling in wild-type and mutant enzymes

His-172 and Tyr-169 are components of a triad in the active site of trimethylamine dehydrogenase (TMADH) comprising Asp-267, His-172, and Tyr-169. Stopped-flow kinetic studies with trimethylamine as substrate have indicated that mutation of His-172 to Gln reduces the limiting rate constant for flavin reduction approximately 10-fold (Basran, J., Sutcliffe, M. J., Hille, R., and Scrutton, N. S. (1999) Biochem. J. 341, 307-314). A kinetic isotope effect (KIE = k(H)/k(D)) accompanies flavin reduction by H172Q TMADH, the magnitude of which varies significantly with solution pH. With trimethylamine, flavin reduction by H172Q TMADH is controlled by a single macroscopic ionization (pK(a) = 6.8 +/- 0.1). This ionization is perturbed (pK(a) = 7.4 +/- 0.1) in reactions with perdeuterated trimethylamine and is responsible for the apparent variation in the KIE with solution pH. At pH 9.5, where the functional group controlling flavin reduction is fully ionized, the KIE is independent of temperature in the range 277-297 K, consistent with vibrationally assisted hydrogen tunneling during breakage of the substrate C-H bond. Y169F TMADH is approximately 4-fold more compromised than H172Q TMADH for hydrogen transfer, which occurs non-classically. Studies with Y169F TMADH suggest partial thermal excitation of substrate prior to hydrogen tunneling by a vibrationally assisted mechanism. Our studies illustrate the varied effects of compromising mutations on tunneling regimes in enzyme molecules.     

Basolateral membrane Na(+)-independent Cl-/HCO3- exchange in the inner stripe of the rabbit outer medullary collecting tubule

The inner stripe of the outer medullary collecting tubule is a major distal nephron segment in urinary acidification. To examine the mechanism of basolateral membrane H+/OH-/HCO3- transport in this segment, cell pH was measured microfluorometrically in the inner stripe of the rabbit outer medullary collecting tubule perfused in vitro using the pH-sensitive fluorescent dye, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein. Decreasing peritubular pH from 7.4 to 6.8 (changing [HCO3-] from 25 to 5 mM) caused a cell acidification of 0.25 +/- 0.02 pH units, while a similar luminal change resulted in a smaller cell acidification of only 0.04 +/- 0.01 pH units. Total replacement of peritubular Cl- with gluconate caused cell pH to increase by 0.18 +/- 0.04 pH units, an effect inhibited by 100 microM peritubular DIDS and independent of Na+. Direct coupling between Cl- and base was suggested by the continued presence of peritubular Cl- removal-induced cell alkalinization under the condition of a cell voltage clamp (K(+)-valinomycin). In addition, 90% of basolateral membrane H+/OH-/HCO3- permeability was inhibited by complete removal of luminal and peritubular Cl-. Peritubular Cl(-)-induced cell pH changes were inhibited two-thirds by removal of exogenous CO2/HCO3- from the system. The apparent Km for peritubular Cl- determined in the presence of 25 mM luminal and peritubular [HCO3-] was 113.5 +/- 14.8 mM. These results demonstrate that the basolateral membrane of the inner stripe of the outer medullary collecting tubule possesses a stilbene-sensitive Cl-/HCO3- exchanger which mediates 90% of basolateral membrane H+/OH-/HCO3- permeability and may be regulated by physiologic Cl- concentrations.     

Vanadyl(IV) binding to mammalian ferritins. An EPR study aided by site-directed mutagenesis

During its metabolism, vanadium is known to become associated with the iron storage protein, ferritin. To elucidate probable vanadium binding sites on the protein, VO2+ binding to mammalian ferritins was studied using site-directed mutagenesis and EPR spectroscopy. VO2+-apoferritin EPR spectra of human H-chain (100% H), L-chain (100% L), horse spleen (84% L, 16% H) and sheep spleen (45% L, 55% H) ferritins revealed the presence of alpha and beta VO2+ species in all the proteins, implying that the ligands for these species are conserved between the H- and L-chains. The alpha species is less stable than the beta species and decreases with increasing pH, demonstrating that the two species are not pH-related, a result contrary to earlier proposals. EPR spectra of site-directed HuHF variants of several residues conserved in H- and L-chain ferritins (Asp-131, Glu-134, His-118 and His-128) suggest that His-118 near the outer opening of the three-fold channel is probably a ligand for VO2+ and is responsible for the beta signals in the EPR spectrum. The data indicate that VO2+ does not bind to the Asp-131 and Glu-134 residues within the three-fold channels nor does it bind at the ferroxidase site residues Glu-62 or His-65 or at the putative nucleation site residues Glu-61,64,67. While the ferroxidase site is not a site for VO2+ binding, mutation of residues Glu-62 and His-65 of this site to Ala affects VO2+ binding at His-118, located some 17 A away. Thus, VO2+ spin probe studies provide a window on structural changes in ferritin not seen in most previous work and indicate that long-range effects caused by point mutations must be carefully considered when drawing conclusions from mutagenesis studies of the protein.     

Nuclear-magnetic-resonance study of the histidine residues of S-peptide and S-protein and kinetics of 1H-2H exchange of ribonuclease A.

1H NMR spectroscopy at 100 MHz was used to determine the first-order rate constants for the 1H-2H exchange of the H-2 histidine resonances of RNase-A in 2H2O at 35 degrees C and pH meter readings of 7, 9, 10 and 10.5. Prolonged exposure in 2H2O at 35 degrees C and pH meter reading 11 caused irreversible denaturation of RN-ase-A. The rate constants at pH 7 and 9 agreed reasonably well with those obtained in 1H-3H exchange experiments by Ohe, J., Matsuo, H., Sakiyama, F. and Narita, K. [J. Biochem, (Tokyo) 75, 1197-1200 (1974)]. The rate data obtained by various authors is summarised and the reasons for the poor agreement between the data is discussed. The first-order rate constant for the exchange of His-48 increases rapidly from near zero at pH 9 (due to its inaccessibility to solvent) with increase of pH to 10.5 The corresponding values for His-119 show a decrease and those for His-12 a small increase over the same pH range. These changes are attributed to a conformational change in the hinge region of RNase-A (probably due to the titration of Tyr-25) which allows His-48 to become accessible to solvent. 1H NMR spectra of S-protein and S-peptide, and of material partially deuterated at the C-2 positions of the histidine residues confirm the reassignment of the histidine resonances of RNase-A [Bradbury, J. H. & Teh, J. S. (1975) Chem. Commun., 936-937]. The chemical shifts of the C-2 and C-4 protons of histidine-12 of S-peptide are followed as a function of pH and a pK' value of 6.75 is obtained. The reassignment of the three C-2 histidine resonances of S-protein is confirmed by partial deuteration studies. The pK' values obtained from titration of the H-2 resonances of His-48, His-105 and His-119 are 5.3, 6.5 and 6.0, respectively. The S-protein is less stable to acid than RNase-A since the former, but not the latter, shows evidence of reversible denaturation at pH 3 and 26 degrees C. His-48 in S-protein titrates normally and has a lower pK than in RN-ase-A probably because of the absence of Asp-14, which in RN-ase-A forms a a hydrogen bond with His-48 and causes it to be inaccessible to solvent, at pH values below 9.     

Hemes and hemeproteins, 2: The pH-dependent equilibria of microperoxidase-8 and characterization of the coordination sphere of Fe(III)

Titration of the monomeric heme octapeptide from horse heart cytochrome c, microperoxidase-8 (MP-8) from pH 1 to pH 13 in 20% (v/v) methanol-water solutions, mu = 0.1, at 25 degrees C shows three reversible concentration-independent pKs (4.43 +/- 0.09; 8.90 +/- 0.03; 10.48 +/- 0.09) which are ascribed to successive proton loss from the conjugate acid of His (and its coordination to Fe(III)), bound H2O, and from bound His to form an imidazolate complex, respectively. The equilibrium constant for coordination of imidazole between pH 5.5 and 7.0 is independent of pH (logK = 4.45) which proves that His-18 is coordinated to Fe(III) in aqueous solution.     

Hemes and hemoproteins. 3. The reaction of microperoxidase-8 with cyanide: comparison with aquocobalamin and hemoproteins

The monomeric heme octapeptide from cytochrome c, microperoxidase-8, (MP-8), coordinates CN- with log K = 7.55 +/- 0.04 at 25 degrees C in 20% (v/v) aqueous methanol. Log K values are independent of pH between 6 and 9. A spectrophotometric titration of cyanoMP-8 between pH 5.5 and 13.8 gave a single pKa greater than or equal to 13.5 ascribed to ionization of the proximal His ligand. A study of the kinetics of the reaction of MP-8 with cyanide between pH 5.5 and 12, at 25 degrees C and mu = 0.1, indicates that formation of cyanoMP-8 occurs via three routes: attack of CN- on Fe(III) (k1 = 6.0 +/- 0.3 X 10(5) M-1 sec-1); attack of HCN on Fe(III) (k2 = 4.8 +/- 2.0 X 10(3) M-1 sec-1), followed by deprotonation and isomerization to form the C-bound species; and displacement of OH- by CN- when the proximal His ligand is ionized (k5 = 1.8 +/- 0.1 X 10(5) M-1 sec-1). These results are compared with available data for the reaction of cyanide with aquocobalamin and with various hemoproteins.     

Water penetration and binding to ferric myoglobin

Flash photolysis investigations of horse heart metmyoglobin bound with NO (Mb(3+)NO) reveal the kinetics of water entry and binding to the heme iron. Photodissociation of NO leaves the sample in the dehydrated Mb(3+) (5-coordinate) state. After NO photolysis and escape, a water molecule enters the heme pocket and binds to the heme iron, forming the 6-coordinate aquometMb state (Mb(3+)H2O). At longer times, NO displaces the H2O ligand to reestablish equilibrium. At 293 K, we determine a value k(w) approximately 5.7 x 10(6) s(-1) for the rate of H2O binding and estimate the H2O dissociation constant as 60 mM. The Arrhenius barrier height H(w) = 42 +/- 3 kJ/mol determined for H2O binding is identical to the barrier for CO escape after photolysis of Mb(2+)CO, within experimental uncertainty, consistent with a common mechanism for entry and exit of small molecules from the heme pocket. We propose that both processes are gated by displacement of His-64 from the heme pocket. We also observe that the bimolecular NO rebinding rate is enhanced by 3 orders of magnitude both for the H64L mutant, which does not bind water, and for the H64G mutant, where the bound water is no longer stabilized by hydrogen bonding with His-64. These results emphasize the importance of the hydrogen bond in stabilizing H2O binding and thus preventing NO scavenging by ferric heme proteins at physiological NO concentrations.     

Identification of the proximal ligand His-20 in heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Oxidative cleavage of the heme macrocycle does not require the proximal histidine

The coordination and spin-state of the Corynebacterium diphtheriae heme oxygenase (Hmu O) and the proximal Hmu O H20A mutant have been characterized by UV-visible and resonance Raman (RR) spectrophotometry. At neutral pH the ferric heme-Hmu O complex is a mixture of six-coordinate high spin and six-coordinate low spin species. Changes in the UV-visible and high frequency RR spectra are observed as a function of pH and temperature, with the six-coordinate high spin species being converted to six-coordinate low spin. The low frequency region of the ferrous RR spectrum identified the proximal ligand to the heme as a neutral imidazole with a Fe-His stretching mode at 222 cm(-1). The RR characterization of the heme-CO complex in wt-Hmu O confirms that the proximal imidazole is neither ionized or strongly hydrogen-bonded. Based on sequence identity with the mammalian enzymes the proximal ligand in HO-1 (His-25) and HO-2 (His-45) is conserved (His-20) in the bacterial enzyme. Site-specific mutagenesis identified His-20 as the proximal mutant based on electronic and resonance Raman spectrophotometric analysis. Titration of the heme-Hmu O complex with imidazole restored full catalytic activity to the enzyme, and the coordination of imidazole to the heme was confirmed by RR. However, in the absence of imidazole, the H20A Hmu O mutant was found to catalyze the initial alpha-meso-hydroxylation of the heme. The product of the aerobic reaction was determined to be ferrous verdoheme. Hydrolytic conversion of the verdoheme product to biliverdin concluded that oxidative cleavage of the porphyrin macrocycle was specific for the alpha-meso-carbon. The present data show that, in marked contrast to the human HO-1, the proximal ligand is not essential for the initial alpha-meso-hydroxylation of heme in the C. diphtheriae heme oxygenase-catalyzed reaction.     

An electrical and structural characterization of H+/OH- currents in phospholipid vesicles

Paramagnetic amphiphiles have been utilized to measure and characterize electrogenic H+/OH- ion transport in a series of model membrane systems. Membrane conductivity to H+/OH- ions varies with the method of vesicle preparation and with the level of saturation of the membrane phospholipid. Small sonicated vesicles have the lowest conductivities by approximately an order of magnitude compared to reverse-phase or ether-injection vesicle systems. This conductivity is particularly sensitive to the presence of polyunsaturated lipids in the vesicle membrane. The current-delta pH dependence of the H+/OH- conductivity shows a nonideal behavior and renders the phenomenological membrane permeability dependent upon the experimental value of delta pH that is chosen. These factors can account for much, if not all, of the variability in the published values for the H+/OH- permeability of model membranes. A procedure has been developed to establish and estimate changes in the dipole potential of vesicle bilayers. Using this method, we demonstrate that H+/OH- currents are insensitive to alterations in the membrane dipole field, a result that suggests that these currents are not rate limited by diffusion over simple electrostatic barriers in the membrane interior. In addition, conduction in D2O has been examined, and we find that there is little difference in the magnitudes of D+/OD- currents compared to H+/OH- currents in vesicle systems.     

Oxidation of anthracycline anticancer agents by the peroxidase mimic microperoxidase 11 and hydrogen peroxide

The interaction of two clinically important anticancer agents doxorubicin (DXR) and daunorubicin (DNR) and the DNR analog 5-iminodaunorubicin (5IDNR) with the model mammalian peroxidase microperoxidase 11 (MP11) and H(2)O(2) has been investigated using spectrophotometric and EPR techniques. We demonstrate that DNR, DXR, and 5IDNR undergo irreversible oxidation by MP11/H(2)O(2), forming colorless products in both phosphate buffer pH 7.0 and in phosphate buffer pH 7.0/MeOH mixture (1:1 vol/vol), suggesting an extensive modification of the compounds' chromophores. The initial rate of the anthracyclines' oxidation is independent of anthracycline concentrations, but is linearly dependent on [H(2)O(2)](i) at constant [MP11](i) (and vice versa), indicating that the reaction is zero order in [anthracycline], first order with respect to [H(2)O(2)] and [MP11], and second order overall. Based on data obtained using DNR, DXR, 5IDNR, and p-hydroquinone k(2app), the apparent second order rate constant for the formation of a reactive intermediate from MP11 and H(2)O(2) (an analog of peroxidase compound I) has been determined to be in the range of (2.51-5.11) x 10(3) M(-1) s(-1) in both solvent systems. EPR studies show that oxidation of DNR, DXR, or 5IDNR with MP11/H(2)O(2) generates free radicals, suggesting that the reaction may be a one-electron process. This study also shows that 5IDNR, but not DNR or DXR, efficiently protects MP11 heme against degradation by H(2)O(2). Our overall conclusion is that MP11 is an effective catalyst of oxidation of anthracyclines by H(2)O(2). Given that, at sites of inflammation or cancer, the anthracyclines can colocalize with peroxidases, protein degradation products, and with H(2)O(2), peroxidation could be one possible fate of these anticancer agents in vivo.     

Direct probing of copper active site and free radical formed during bicarbonate-dependent peroxidase activity of bovine and human copper, zinc-superoxide dismutases. Low-temperature electron paramagnetic resonance and electron nuclear double resonance studies

Using X-band electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy at liquid helium temperatures, the Cu(II) coordination geometry at the active site of bovine and human copper,zinc-superoxide dismutases (bSOD1 and hSOD1) treated with H(2)O(2) and bicarbonate (HCO(3)(-)) was examined. The time course EPR of wild type human SOD1 (WT hSOD1), W32F hSOD1 mutant (tryptophan 32 substituted with phenylalanine), and bSOD1 treated with H(2)O(2) and HCO(3)(-) shows an initial reduction of active site Cu(II) to Cu(I) followed by its oxidation back to Cu(II) in the presence of H(2)O(2). However, HCO(3)(-) induced a Trp-32-derived radical from WT hSOD1 but not from bSOD1. The mutation of Trp-32 by phenylalanine totally eliminated the Trp-32 radical signal generated from W32F hSOD1 treated with HCO(3)(-) and H(2)O(2). Further characterization of the free radical was performed by UV irradiation of WT hSOD1 and bSOD1 that generated tryptophanyl and tyrosyl radicals. Both proton ((1)H) and nitrogen ((14)N) ENDOR studies of bSOD1 and hSOD1 in the presence of H(2)O(2) revealed a change in the geometry of His-46 (or His-44) and His-48 (or His-46) coordinated to Cu(II) at the active site of WT hSOD1 and bSOD1, respectively. However, in the presence of HCO(3)(-) and H(2)O(2), both (1)H and (14)N ENDOR spectra were almost identical to those derived from native bSOD1. We conclude that HCO(3)(-)-derived oxidant does not alter significantly the Cu(II) active site geometry and histidine coordination to Cu(II) in SOD1 as does H(2)O(2) alone; however, the oxidant derived from HCO(3)(-) (i.e. carbonate anion radical) reacts with surface-associated Trp-32 in hSOD1 to form the corresponding radical.     

Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154-->Cys forms of yeast xylitol dehydrogenase

Co-ordination of catalytic Zn2+ in sorbitol/xylitol dehydrogenases of the medium-chain dehydrogenase/reductase superfamily involves direct or water-mediated interactions from a glutamic acid residue, which substitutes a homologous cysteine ligand in alcohol dehydrogenases of the yeast and liver type. Glu154 of xylitol dehydrogenase from the yeast Galactocandida mastotermitis (termed GmXDH) was mutated to a cysteine residue (E154C) to revert this replacement. In spite of their variable Zn2+ content (0.10-0.40 atom/subunit), purified preparations of E154C exhibited a constant catalytic Zn2+ centre activity (kcat) of 1.19+/-0.03 s(-1) and did not require exogenous Zn2+ for activity or stability. E154C retained 0.019+/-0.003% and 0.74+/-0.03% of wild-type catalytic efficiency (kcat/K(sorbitol)=7800+/-700 M(-1) x s(-1)) and kcat (=161+/-4 s(-1)) for NAD+-dependent oxidation of sorbitol at 25 degrees C respectively. The pH profile of kcat/K(sorbitol) for E154C decreased below an apparent pK of 9.1+/-0.3, reflecting a shift in pK by about +1.7-1.9 pH units compared with the corresponding pH profiles for GmXDH and sheep liver sorbitol dehydrogenase (termed slSDH). The difference in pK for profiles determined in 1H2O and 2H2O solvent was similar and unusually small for all three enzymes (approximately +0.2 log units), suggesting that the observed pK in the binary enzyme-NAD+ complexes could be due to Zn2+-bound water. Under conditions eliminating their different pH-dependences, wild-type and mutant GmXDH displayed similar primary and solvent deuterium kinetic isotope effects of 1.7+/-0.2 (E154C, 1.7+/-0.1) and 1.9+/-0.3 (E154C, 2.4+/-0.2) on kcat/K(sorbitol) respectively. Transient kinetic studies of NAD+ reduction and proton release during sorbitol oxidation by slSDH at pH 8.2 show that two protons are lost with a rate constant of 687+/-12 s(-1) in the pre-steady state, which features a turnover of 0.9+/-0.1 enzyme equivalents as NADH was produced with a rate constant of 409+/-3 s(-1). The results support an auxiliary participation of Glu154 in catalysis, and possible mechanisms of proton transfer in sorbitol/xylitol dehydrogenases are discussed.