Steady-state and transient kinetics of Escherichia coli nitric-oxide dioxygenase (flavohemoglobin). The B10 tyrosine hydroxyl is essential for dioxygen binding and catalysis

Escherichia coli expresses an inducible flavohemoglobin possessing robust NO dioxygenase activity. At 37 degrees C, the enzyme shows a maximal turnover number (V(max)) of 670 s(-1) and K(m) values for NADH, NO, and O(2) equal to 4.8, 0.28, and approximately 100 microM, respectively. Individual reduction, ligand binding, and NO dioxygenation reactions were examined at 20 degrees C, where V(max) is approximately 94 s(-1). Reduction by NADH occurs in two steps. NADH reduces bound FAD with a rate constant of approximately 15 microM(-1) s(-1), and heme iron is reduced by FADH(2) with a rate constant of 150 s(-1). Dioxygen binds tightly to reduced flavohemoglobin, with association and dissociation rate constants equal to 38 microM(-1) s(-1) and 0.44 s(-1), respectively, and the oxygenated flavohemoglobin dioxygenates NO to form nitrate. NO also binds reversibly to reduced flavohemoglobin in competition with O(2), dissociates slowly, and inhibits NO dioxygenase activity at [NO]/[O(2)] ratios of 1:100. Replacement of the heme pocket B10 tyrosine with phenylalanine increases the O(2) dissociation rate constant approximately 80-fold and reduces NO dioxygenase activity approximately 30-fold, demonstrating the importance of the tyrosine hydroxyl for O(2) affinity and NO scavenging activity. At 37 degrees C, V(max)/K(m)(NO) is 2,400 microM(-1) s(-1), demonstrating that the enzyme is extremely efficient at converting toxic NO into nitrate under physiological conditions.     

Nitric oxide dioxygenase function and mechanism of flavohemoglobin, hemoglobin, myoglobin and their associated reductases

Microbial flavohemoglobins (flavoHbs) and hemoglobins (Hbs) show large *NO dioxygenation rate constants ranging from 745 to 2900 microM(-1) s(-1) suggesting a primal *NO dioxygenase (NOD) (EC 1.14.12.17) function for the ancient Hb superfamily. Indeed, modern O2-transporting and storing mammalian red blood cell Hb and related muscle myoglobin (Mb) show vestigial *NO dioxygenation activity with rate constants of 34-89 microM(-1) s(-1). In support of a NOD function, microbial flavoHbs and Hbs catalyze O2-dependent cellular *NO metabolism, protect cells from *NO poisoning, and are induced by *NO exposures. Red blood cell Hb, myocyte Mb, and flavoHb-like activities metabolize *NO in the vascular lumen, muscle, and other mammalian cells, respectively, decreasing *NO signalling and toxicity. HbFe(III)-OO*, HbFe(III)-OONO and protein-caged [HbFe(III)-O**NO2] are proposed intermediates in a reaction mechanism that combines both O-atoms of O2 with *NO to form nitrate and HbFe(III). A conserved Hb heme pocket structure facilitates the dioxygenation reaction and efficient turnover is achieved through the univalent reduction of HbFe(III) by associated reductases. High affinity flavoHb and Hb heme ligands, and other inhibitors, may find application as antibiotics and antitumor agents that enhance the toxicity of immune cell-derived *NO or as vasorelaxants that increase *NO signalling.     

Dos, a heme-binding PAS protein from Escherichia coli, is a direct oxygen sensor

A direct sensor of O(2), the Dos protein, has been found in Escherichia coli. Previously, the only biological sensors known to respond to O(2) by direct and reversible binding were the FixL proteins of Rhizobia. A heme-binding region in Dos is 60% homologous to the O(2)-sensing PAS domain of the FixL protein, but the remainder of Dos does not resemble FixL. Specifically, the C-terminal domain of Dos, presumed to be a regulatory partner that couples to its heme-binding domain, is not a histidine kinase but more closely resembles a phosphodiesterase. The absorption spectra of Dos indicate that both axial positions of the heme iron are coordinated to side chains of the protein. Nevertheless, O(2) and CO bind to Dos with K(d) values of 13 and 10 microM, respectively, indicating a strong discrimination against CO binding. Association rate constants for binding of O(2) (3 mM(-)(1) s(-)(1)), CO (1 mM(-)(1) s(-)(1)) and even NO (2 mM(-)(1) s(-)(1)) are extraordinarily low and very similar. Displacement of an endogenous ligand, probably Met 95, from the heme iron in Dos triggers a conformational change that alters the activity of the enzymatic domain. This sensing mechanism differs from that of FixL but resembles that of the CO sensor CooA of Rhodospirillum rubrum. Overall the results provide evidence for a heme-binding subgroup of PAS-domain proteins whose working range, signaling mechanisms, and regulatory partners can vary considerably.     

A "sliding scale rule" for selectivity among NO, CO, and O₂ by heme protein sensors

Selectivity among NO, CO, and O? is crucial for the physiological function of most heme proteins. Although there is a million-fold variation in equilibrium dissociation constants (K(D)), the ratios for NO:CO:O? binding stay roughly the same, 1:~10(3):~10(6), when the proximal ligand is a histidine and the distal site is apolar. For these proteins, there is a "sliding scale rule" for plots of log(K(D)) versus ligand type that allows predictions of K(D) values if one or two are missing. The predicted K(D) for binding of O?to Ns H-NOX coincides with the value determined experimentally at high pressures. Active site hydrogen bond donors break the rule and selectively increase O? affinity with little effect on CO and NO binding. Strong field proximal ligands such as thiolate, tyrosinate, and imidazolate exert a "leveling" effect on ligand binding affinity. The reported picomolar K(D) for binding of NO to sGC deviates even more dramatically from the sliding scale rule, showing a NO:CO K(D) ratio of 1:~10(8). This deviation is explained by a complex, multistep process, in which an initial low-affinity hexacoordinate NO complex with a measured K(D) of ≈54 nM, matching that predicted from the sliding scale rule, is formed initially and then is converted to a high-affinity pentacoordinate complex. This multistep six-coordinate to five-coordinate mechanism appears to be common to all NO sensors that exclude O? binding to capture a lower level of cellular NO and prevent its consumption by dioxygenation.     

Dioxygen-dependent metabolism of nitric oxide in mammalian cells.

Steady-state gradients of NO within tissues and cells are controlled by rates of NO synthesis, diffusion, and decomposition. Mammalian cells and tissues actively decompose NO. Of several cell lines examined, the human colon CaCo-2 cell produces the most robust NO consumption activity. Cellular NO metabolism is mostly O2-dependent, produces near stoichiometric NO3-, and is inhibited by the heme poisons CN-, CO (K(I) approximately 3 microM), phenylhydrazine, and NO and the flavoenzyme inhibitor diphenylene iodonium. NO consumption is saturable by O2 and NO and shows apparent K(M) values for O2 and NO of 17 and 0.2 microM, respectively. Mitochondrial respiration, O2*-, and H2O2 are neither sufficient nor necessary for O2-dependent NO metabolism by cells. The existence of an efficient mammalian heme and flavin-dependent NO dioxygenase is suggested. NO dioxygenation protects the NO-sensitive aconitases, cytochrome c oxidase, and cellular respiration from inhibition, and may serve a dual function in cells by limiting NO toxicity and by spatially coupling NO and O2 gradients.     

Encapsulation of hemoglobin inside liposomes surface conjugated with poly(ethylene glycol) attenuates their reactions with gaseous ligands and regulates nitric oxide dependent vasodilation

Acellular hemoglobin (Hb)-based O2 carriers (HBOCs) are being investigated as red blood cell (RBC) substitutes for use in transfusion medicine. However, commercial acellular HBOCs elicit both vasoconstriction and systemic hypertension which hampers their clinical use. In this study, it is hypothesized that encapsulation of Hb inside the aqueous core of liposomes should regulate the rates of NO dioxygenation and O2 release, which should in turn regulate its vasoactivity. To test this hypothesis, poly(ethylene glycol) (PEG) conjugated liposome-encapsulated Hb (PEG-LEHs) dispersions were prepared using human and bovine Hb. In this study, the rate constants for O2 dissociation, CO association, and NO dioxygenation were measured for free Hb and PEG-LEH dispersions using stopped-flow UV-visible spectroscopy, while vasoactivity was assessed in rat aortic ring strips using both endogenous and exogenous sources of NO. It was observed that PEG-LEH dispersions had lower O2 release and NO dioxygenation rate constants compared with acellular Hbs. However, no difference was observed in the CO association rate constants between free Hb and PEG-LEH dispersions. Furthermore, it was observed that Hb encapsulation inside vesicles prevented Hb dependent inhibition of NO-mediated vasodilation. In addition, the magnitude of the vasoconstrictive effects of Hb and PEG-LEH dispersions correlated with their respective rates of NO dioxygenation and O2 release. Overall, this study emphasizes the pivotal role Hb encapsulation plays in regulating gaseous ligand binding/release kinetics and the vasoactivity of Hb.     

Structure of Ralstonia eutropha flavohemoglobin in complex with three antibiotic azole compounds

Flavohemoglobins (flavoHbs) are enzymes that operate primarily as nitric oxide dioxygenases and shuttle thereby electrons among NAD(P)H, FAD, heme, and a ligated redox-active substrate such as O(2). They function in the bacterial defense against nitrosative stress and are therefore considered as targets for new antibiotic drugs. Recently, azole derivatives were proven to be attractive nitric oxide dioxygenase inhibitors, and to explore their binding characteristics, we determined the X-ray structure of the flavoHb from Ralstonia eutropha in a complex with miconazole (FHP(M)), econazole (FHP(E)), and ketoconazole (FHP(K)). In agreement with UV-vis spectroscopic data, one azole compound binds inside the distal heme pocket and ligates to the heme iron by its imidazole substituent. The two additional substituents, mostly chlorinated phenyl groups, form a series of van der Waals contacts with the protein matrix. Both interactions explain their high affinity for flavoHbs, the binding constants being 2.6, 1.2, and 11.6 μM for miconazole, econazole, and ketoconazole, respectively. The FHP(M) and FHP(Lip) (flavoHbs originally loaded with a phospholipid) structures share an "open" state and the FHP(E) and FHP(K) structures a "closed" state. Although the azole compounds were able to push the lipid out of its binding site, a fatty acid fragment is still bound inside the heme pocket of FHP(E) and FHP(K) and dictates the state of the protein. The ligand-induced open-to-closed transition involves a reorientation of the NADH domain accompanied by conformational changes in the C-terminal arm, helix E, and the CE loop resulting in an encapsulation of the heme-binding pocket. Implications of the observed open-to-closed process on the catalytic cycle are discussed.     

Binding of oxygen and carbon monoxide to a heme-regulated phosphodiesterase from Escherichia coli. Kinetics and infrared spectra of the full-length wild-type enzyme, isolated PAS domain, and Met-95 mutants

The heme-regulated phosphodiesterase, Ec DOS, is a redox sensor that uses the heme in its PAS domain to regulate catalysis. The rate of O(2) association (k(on)) with full-length Ec DOS is extremely slow at 0.0019 microM(-1) s(-1), compared with >9.5 microM(-1) s(-1) for 6-coordinated globin-type hemoproteins, as determined by the stopped-flow method. This rate is dramatically increased (up to 16-fold) in the isolated heme-bound PAS domain. Dissociation constants (K(d)) calculated from the kinetic parameters are 340 and 20 microm for the full-length wild-type enzyme and its isolated PAS domain, respectively. Mutations at Met-95 in the isolated PAS domain, which may be a heme axial ligand in the Fe(II) complex, lead to a further increase in the k(on) value by more than 30-fold, and consequently, a decrease in the K(d) value to less than 1 microM. The k(on) value for CO binding to the full-length wild-type enzyme is also very low (0.00081 microM(-1) s(-1)). The kinetics of CO binding to the isolated PAS domain and its mutants are similar to those observed for O(2). However, the K(d) values for CO are considerably lower than those for O(2).     

Oxygen dependence of mitochondrial nitric oxide synthase activity

The effect of O(2) concentration on mitochondrial nitric oxide synthase (mtNOS) activity and on O(2)(-) production was determined in rat liver, brain, and kidney submitochondrial membranes. The K(mO(2)) for mtNOS were 40, 73, and 37 microM O(2) and the V(max) were 0.51, 0.49, and 0.42 nmol NO/minmg protein for liver, brain, and kidney mitochondria, respectively. The rates of O(2)(-) production, 0.5-12.8 nmol O(2)(-)/minmg protein, depended on O(2) concentration up to 1.1mM O(2). Intramitochondrial NO, O(2)(-), and ONOO(-) steady-state concentrations were calculated for the physiological level of 20 microM O(2); they were 20-39 nM NO, 0.17-0.33 pM O(2)(-), and 0.6-2.2 nM ONOO(-) for the three organs. These levels establish O(2)/NO ratios of 513-1000 that correspond to physiological inhibitions of cytochrome oxidase by intramitochondrial NO of 16-25%. The production of NO by mtNOS appears as a regulatory process that modulates mitochondrial oxygen uptake and cellular energy production.     

The effect of temperature on C(4)-type leaf photosynthesis parameters

C(4)-type photosynthesis is known to vary with growth and measurement temperatures. In an attempt to quantify its variability with measurement temperature, the photosynthetic parameters - the maximum catalytic rate of the enzyme ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) (V(cmax)), the maximum catalytic rate of the enzyme phosphoenolpyruvate carboxylase (PEPC) (V(pmax)) and the maximum electron transport rate (J(max)) - were examined. Maize plants were grown in climatic-controlled phytotrons, and the curves of net photosynthesis (A(n)) versus intercellular air space CO(2) concentrations (C(i)), and A(n) versus photosynthetic photon flux density (PPFD) were determined over a temperature range of 15-40 degrees C. Values of V(cmax), V(pmax) and J(max) were computed by inversion of the von Caemmerer & Furbank photosynthesis model. Values of V(pmax) and J(max) obtained at 25 degrees C conform to values found in the literature. Parameters for an Arrhenius equation that best fits the calculated values of V(cmax), V(pmax) and J(max) are then proposed. These parameters should be further tested with C(4) plants for validation. Other model key parameters such as the mesophyll cell conductance to CO(2) (g(i)), the bundle sheath cells conductance to CO(2) (g(bs)) and Michaelis-Menten constants for CO(2) and O(2) (K(c), K(p) and K(o)) also vary with temperature and should be better parameterized.     

Stopped-flow and steady-state study of the diphenolase activity of mushroom tyrosinase

The reaction of mushroom (Agaricus bisporus) tyrosinase with dioxygen in the presence of several o-diphenolic substrates has been studied by steady-state and transient-phase kinetics in order to elucidate the rate-limiting step and to provide new insights into the mechanism of oxidation of these substrates. A kinetic analysis has allowed for the first time the determination of individual rate constants for several of the partial reactions that comprise the catalytic cycle. Mushroom tyrosinase rapidly reacts with dioxygen with a second-order rate constant k(+8) = 2.3 x 10(7) M(-)(1) s(-)(1), which is similar to that reported for hemocyanins [(1.3 x 10(6))-(5.7 x 10(7)) M(-)(1) s(-)(1)]. Deoxytyrosinase binds dioxygen reversibly at the binuclear Cu(I) site with a dissociation constant K(D)(O)()2 = 46.6 microM, which is similar to the value (K(D)(O)()2 = 90 microM) reported for the binding of dioxygen to Octopus vulgaris deoxyhemocyanin [Salvato et al. (1998) Biochemistry 37, 14065-14077]. Transient and steady-state kinetics showed that o-diphenols such as 4-tert-butylcatechol react significantly faster with mettyrosinase (k(+2) = 9.02 x 10(6) M(-)(1) s(-)(1)) than with oxytyrosinase (k(+6) = 5.4 x 10(5) M(-)(1) s(-)(1)). This difference is interpreted in terms of differential steric and polar effects that modulate the access of o-diphenols to the active site for these two forms of the enzyme. The values of k(cat) for several o-diphenols are also consistent with steric and polar factors controlling the mobility, orientation, and thence the reactivity of substrates at the active site of tyrosinase.     

Sublethal effects of copper on the freshwater crab Potamonautes warreni

The mass specific rates of oxygen consumption (M (O(2)) M(b)(-1)), ammonia excretion (M (NH(4)-N) M(b)(-1)) and carbon dioxide production (M (CO(2)) M(b)(-1)) were measured after 7, 14 and 21 days exposure of adult Potamonautes warreni to a sublethal concentration of 1.0 mg Cu l(-1) (15.75 micromol l(-1)). Under control (non-copper-exposed) conditions M (O(2)) M(b)(-1) was 35.7+/-8.5 micromol kg(-1)min(-1) (mean+/-S.D.), M (NH(4)-N) M(b)(-1) 2.92+/-0.26 micromol kg(-1)min(-1) and M (CO(2)) M(b)(-1) 25.6+/-9.0 micromol kg(-1)min(-1). The oxygen:nitrogen (O:N) ratio and respiratory quotient (RQ) were 24.5+/-3.0 and 0.80+/-0.06, respectively. M (O(2)) M(b)(-1) of copper-exposed crabs showed a significant increase after 7 and 14 days, but decreased significantly by 40% after 21 days. From the increased O:N ratio and RQ below 0.7, it is clear that crabs exposed to 1 mg Cul(-1) metabolize lipids during the entire 21-day exposure period. Free fatty acids in the midgut gland were determined by GC-MS, and showed increases of up to 600% in some C14 to C18 fatty acids. It is proposed that the excess lipids inhibit the pyruvate dehydrogenase complex, leading to the acceleration of the gluco- and glyco-neogenic pathways. Increased glyconeogenesis results in elevated glycogen concentrations in all tissues after 21 days. Experiments on acutely exposed P. warreni show increased incorporation of 14C-labelled lactate into glycogen.     

The rate of nitrite reduction in leaves as indicated by O₂ and CO₂ exchange during photosynthesis

Light response (at 300 ppm CO(2) and 10-50 ppm O(2) in N(2)) and CO(2) response curves [at absorbed photon fluence rate (PAD) of 550 μmol m(-2) s(-1)] of O(2) evolution and CO(2) uptake were measured in tobacco (Nicotiana tabacum L.) leaves grown on either NO(3)(-) or NH(4)(+) as N source and in potato (Solanum tuberosum L.), sorghum (Sorghum bicolor L. Moench), and amaranth (Amaranthus cruentus L.) leaves grown on NH(4)NO(3). Photosynthetic O(2) evolution in excess of CO(2) uptake was measured with a stabilized zirconia O(2) electrode and an infrared CO(2) analyser, respectively, and the difference assumed to represent the rate of electron flow to acceptors alternative to CO(2), mainly NO(2)(-), SO(4)(2-), and oxaloacetate. In NO(3)(-)-grown tobacco, as well as in sorghum, amaranth, and young potato, the photosynthetic O(2)-CO(2) flux difference rapidly increased to about 1 μmol m(-2) s(-1) at very low PADs and the process was saturated at 50 μmol quanta m(-2) s(-1). At higher PADs the O(2)-CO(2) flux difference continued to increase proportionally with the photosynthetic rate to a maximum of about 2 μmol m(-2) s(-1). In NH(4)(+)-grown tobacco, as well as in potato during tuber filling, the low-PAD component of surplus O(2) evolution was virtually absent. The low-PAD phase was ascribed to photoreduction of NO(2)(-) which successfully competes with CO(2) reduction and saturates at a rate of about 1 μmol O(2) m(-2) s(-1) (9% of the maximum O(2) evolution rate). The high-PAD component of about 1 μmol O(2) m(-2) s(-1), superimposed on NO(2)(-) reduction, may represent oxaloacetate reduction. The roles of NO(2)(-), oxaloacetate, and O(2) reduction in the regulation of ATP/NADPH balance are discussed.     

The expression of a carbon concentrating mechanism in Chlamydomonas acidophila under variable phosphorus, iron, and CO2 concentrations

The CO(2) acquisition was analyzed in Chlamydomonas acidophila at pH 2.4 in a range of medium P and Fe concentrations and at high and low CO(2) condition. The inorganic carbon concentrating factor (CCF) was related to cellular P quota (Q(p)), maximum CO(2)-uptake rate by photosynthesis (V(max,O2)), half saturation constant for CO(2) uptake (K(0.5)), and medium Fe concentration. There was no effect of the medium Fe concentration on the CCF. The CCF increased with increasing Q(p) in both high and low CO(2) grown algae, but maximum Q(p) was 6-fold higher in the low CO(2) cells. In high CO(2) conditions, the CCF was low, ranging between 0.8 and 3.5. High CCF values up to 9.1 were only observed in CO(2)-limited cells, but P- and CO(2)-colimited cells had a low CCF. High CCF did not relate with a low K(0.5) as all CO(2)-limited cells had a low K(0.5) (<4 μM CO(2)). High C(i)-pools in cells with high Q(p) suggested the presence of an active CO(2)-uptake mechanism. The CCF also increased with increasing V(max,O2) which reflect an adaptation to the nutrient in highest demand (CO(2)) under balanced growth conditions. It is proposed that the size of the CCF in C. acidophila is more strongly related to porter density for CO(2) uptake (reflected in V(max,O2)) and less- to high-affinity CO(2) uptake (low K(0.5)) at balanced growth. In addition, high CCF can only be realized with high Q(p).     

Interactions of nitric oxide with copper(II) dithiocarbamates in aqueous solution

This is the first report on the formation of air-stable copper nitrosyl complexes. The interaction of nitric oxide, NO, with Cu(DTC)(2).3H(2)O (DTC: dithiocarbamate) and was studied in aqueous solution at pH 7.4 and 293 K. The stability constants were determined from UV-Vis data, using LETAGROP program. The high values obtained, log beta(1)=9.743(5) and log beta(2)=15.44(2) for Cu(ProDTC)(2)-NO, (ProDTC=L-prolinedithiocarbamate) and log beta(1)=8.723(5) and log beta(2)=11.45(2) for Cu(MorDTC)(2)-NO system, (MorDTC=morpholyldithiocarbamate), indicate the formation of two stable nitrosyl complexes, Cu(DTC)(2)NO and Cu(DTC)(2)(NO)(2). Coordinated NO is neither affected by the presence of air nor when the solution is purged with Ar. Cu(MorDTC)(2)NO.3H(2)O was isolated in the solid state and its nuNO (IR) band at 1682 cm(-1), but affected by temperature variations over 333 K.     

Nature of the displaceable heme-axial residue in the EcDos protein,a heme-based sensor from Escherichia coli

The EcDos protein belongs to a group of heme-based sensors that detect their ligands with a heme-binding PAS domain. Among these various heme-PAS proteins, EcDos is unique in having its heme iron coordinated at both axial positions to residues of the protein. To achieve its high affinities for ligands, one of the axial heme-iron residues in EcDos must be readily displaceable. Here we present evidence from mutagenesis, ligand-binding measurements, and magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopies about the nature of the displaceable residue in the heme-PAS domain of EcDos, i.e., EcDosH. The magnetic circular dichroism spectra in the near-infrared region establish histidine-methionine coordination in met-EcDos. To determine whether in deoxy-EcDos coordination of the sixth axial position is also to methionine, methionine 95 was substituted with isoleucine. This substitution caused the ferrous heme iron to change from an exclusively hexacoordinate low-spin form (EcDosH) to an exclusively pentacoordinate high-spin form (M95I EcDosH). This was accompanied by a modest acceleration of the dissociation rates of ligands but a dramatic increase (60-1300-fold) in the association rate constants for binding of O(2), CO, and NO. As a result, the affinity for O(2) was enhanced 10-fold in M95I EcDosH, but the partition constant M = [K(d)(O(2))/K(d)(CO)] between CO and O(2) was raised to about 30 from the extraordinarily low EcDosH value of 1. Thus a major consequence of the increased O(2) affinity of this sensor was the loss of its unusually strong ligand discrimination.     

O2- and alpha-ketoglutarate-dependent tyrosyl radical formation in TauD,an alpha-keto acid-dependent non-heme iron dioxygenase

Taurine/alpha-ketoglutarate dioxygenase (TauD), a non-heme mononuclear Fe(II) oxygenase, liberates sulfite from taurine in a reaction that requires the oxidative decarboxylation of alpha-ketoglutarate (alphaKG). The lilac-colored alphaKG-Fe(II)TauD complex (lambda(max) = 530 nm; epsilon(530) = 140 M(-)(1) x cm(-)(1)) reacts with O(2) in the absence of added taurine to generate a transient yellow species (lambda(max) = 408 nm, minimum of 1,600 M(-)(1) x cm(-)(1)), with apparent first-order rate constants for formation and decay of approximately 0.25 s(-)(1) and approximately 0.5 min(-)(1), that transforms to yield a greenish brown chromophore (lambda(max) = 550 nm, 700 M(-)(1) x cm(-)(1)). The latter feature exhibits resonance Raman vibrations consistent with an Fe(III) catecholate species presumed to arise from enzymatic self-hydroxylation of a tyrosine residue. Significantly, (18)O labeling studies reveal that the added oxygen atom derives from solvent rather than from O(2). The transient yellow species, identified as a tyrosyl radical on the basis of EPR studies, is formed after alphaKG decomposition. Substitution of two active site tyrosine residues (Tyr73 and Tyr256) by site-directed mutagenesis identified Tyr73 as the likely site of formation of both the tyrosyl radical and the catechol-associated chromophore. The involvement of the tyrosyl radical in catalysis is excluded on the basis of the observed activity of the enzyme variants. We suggest that the Fe(IV) oxo species generally proposed (but not yet observed) as an intermediate for this family of enzymes reacts with Tyr73 when substrate is absent to generate Fe(III) hydroxide (capable of exchanging with solvent) and the tyrosyl radical, with the latter species participating in a multistep TauD self-hydroxylation reaction.     

Dissociation constants of protonated methionine species in NaCl media

The sulfur-containing amino acid, methionine, has a role in the physiological environment because of its strong interactions with metals. To understand these interactions of metals with methionine, one needs reliable dissociation constants for the protonated methionine species (NH(3)(+)CH(CH(2)CH(2)SCH(3))COOH; H(2)B(+)). The values of stoichiometric dissociation constants, pK(i)*, for protonated methionine species (H(2)B(+) if H(+)+HB, K(1); HB if H(+)+B(-), K(2)) were determined from potentiometric measurements in NaCl solutions as a function of ionic strength, 0.25-6.0 mol (kg H(2)O)(-1) and temperature (5-45 degrees C). The results were extrapolated to pure water using the Pitzer equations to estimate the activity of H(+), H(2)B(+), HB and B(-) as a function of ionic strength and temperature. The resulting thermodynamic values of K(1) and K(2) were fit to the equations (T/K): ln K(1)=69.0013-3496.58/(T/K)-10.9153 ln (T/K); ln K(2)=116.4162-10638.02/(T/K)-18.0553 ln (T/K) with standard errors of 0.003 and 0.033, respectively, for ln K(1)* and ln K(2)*. Pitzer interaction parameters (lambda(HB-Na) and zeta(HB-Na-Cl)) for the neutral HB were determined from literature data. The Pitzer parameters (beta(0)(H(2)BCl), beta(1)(H(2)BCl) and C(phi)(H(2)BCl)) for the interactions of H(2)B(+) with Cl(-) and Na(+) with and B(2-) (beta(0)(NaB), beta(1)(NaB) and C(phi)(NaB)) were also determined. These coefficients can be used to make reasonable estimates of the activity coefficients of methionine species and the pK(i)(*) for the dissociation of methionine in physiological solutions, composed mostly of NaCl over a wide range of temperature and ionic strength.     

Quantitative comparison of lactose and glucose utilization in Bifidobacterium longum cultures

In batch cultures, Bifidobacterium longum SH2 has a higher final cell concentration and greater substrate consumption when grown on lactose versus glucose. Continuous cultures were used to compare lactose and glucose utilization by B. longum quantitatively. In the continuous culture, the estimated maintenance coefficients (m) were similar when on lactose and glucose; the maximum cell yield coefficient (Y(X/S)(max)) was higher on lactose; and the specific consumption rate of lactose (q(S)) was lower than that of glucose. Assuming that cell growth followed the Monod model, the maximum specific growth rates (mu(max)) and saturation constants (K(S)) in lactose and glucose media were determined using the Hanes-Woolf plots. The respective values were 0.40 h(-)(1) and 78 mg/L for lactose and 0.46 h(-)(1) and 697 mg/L for glucose. The kinetic parameters of the continuous cultures showed that B. longum preferred lactose to glucose, although the specific consumption rate of glucose was higher than that of lactose.     

The mode of decomposition of Angeli's salt (Na2N2O3) and the effects thereon of oxygen,nitrite, superoxide dismutase,and glutathione

The classical view of the aerobic decomposition of Angeli's salt is that it releases NO(2)(-) + NO(-)/HNO the latter then reacting with O(2) to yield ONOO(-). An alternative that has recently been proposed envisions electron transfer to O(2) followed by decomposition to NO(2)(-) + NO. The classical view is now strongly supported by the observation that the rates of decomposition of Angeli's salt under 20% O(2) or 100% O(2) were equal. Moreover, NO(2)(-), which inhibits this decomposition by favoring the back reaction, was more effective in the absence of agents that scavenge NO(-)/HNO. It is thus clear that Angeli's salt is a useful source of NO(-)/HNO for use in defined aqueous systems. The measurements made in the course of this work allowed approximation of the rate constants for the reactions of NO(-)/HNO with NO(2)(-), O(2), glutathione, or Cu, Zn superoxide dismutase. The likelihood of the formation of NO(-)/HNO in vivo is also discussed.