Nitric-oxide dioxygenase activity and function of flavohemoglobins. sensitivity to nitric oxide and carbon monoxide inhibition

Widely distributed flavohemoglobins (flavoHbs) function as NO dioxygenases and confer upon cells a resistance to NO toxicity. FlavoHbs from Saccharomyces cerevisiae, Alcaligenes eutrophus, and Escherichia coli share similar spectra, O(2), NO, and CO binding kinetics, and steady-state NO dioxygenation kinetics. Turnover numbers (V(max)) for S. cerevisiae, A. eutrophus, and E. coli flavoHbs are 112, 290, and 365 NO heme(-1) s(-1), respectively, at 37 degrees C with 200 microm O(2). The K(M) values for NO are low and range from 0.1 to 0.25 microm. V(max)/K(M)(NO) ratios of 900-2900 microm(-1) s(-1) indicate an extremely efficient dioxygenation mechanism. Approximate K(M) values for O(2) range from 60 to 90 microm. NO inhibits the dioxygenases at NO:O(2) ratios of > or =1:100 and makes true K(M)(O(2)) values difficult to determine. High and roughly equal second order rate constants for O(2) and NO association with the reduced flavoHbs (17-50 microm(-1) s(-1)) and small NO dissociation rate constants suggest that NO inhibits the dioxygenase reaction by forming inactive flavoHbNO complexes. Carbon monoxide also binds reduced flavoHbs with high affinity and competitively inhibits NO dioxygenases with respect to O(2) (K(I)(CO) = approximately 1 microm). These results suggest that flavoHbs and related hemoglobins evolved as NO detoxifying components of nitrogen metabolism capable of discriminating O(2) from inhibitory NO and CO.     

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.     

Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis

Microbial xylose reductase, a representative aldo-keto reductase of primary sugar metabolism, catalyzes the NAD(P)H-dependent reduction of D-xylose with a turnover number approximately 100 times that of human aldose reductase for the same reaction. To determine the mechanistic basis for that physiologically relevant difference and pinpoint features that are unique to the microbial enzyme among other aldo/keto reductases, we carried out stopped-flow studies with wild-type xylose reductase from the yeast Candida tenuis. Analysis of transient kinetic data for binding of NAD(+) and NADH, and reduction of D-xylose and oxidation of xylitol at pH 7.0 and 25 degrees C provided estimates of rate constants for the following mechanism: E + NADH right arrow over left arrow E.NADH right arrow over left arrow E.NADH + D-xylose right arrow over left arrow E.NADH.D-xylose right arrow over left arrow E.NAD(+).xylitol right arrow over left arrow E.NAD(+) right arrow over left arrow E.NAD(+) right arrow over left arrow E + NAD(+). The net rate constant of dissociation of NAD(+) is approximately 90% rate limiting for k(cat) of D-xylose reduction. It is controlled by the conformational change which precedes nucleotide release and whose rate constant of 40 s(-)(1) is 200 times that of completely rate-limiting E.NADP(+) --> E.NADP(+) step in aldehyde reduction catalyzed by human aldose reductase [Grimshaw, C. E., et al. (1995) Biochemistry 34, 14356-14365]. Hydride transfer from NADH occurs with a rate constant of approximately 170 s(-1). In reverse reaction, the E.NADH --> E.NADH step takes place with a rate constant of 15 s(-1), and the rate constant of ternary-complex interconversion (3.8 s(-1)) largely determines xylitol turnover (0.9 s(-1)). The bound-state equilibrium constant for C. tenuis xylose reductase is estimated to be approximately 45 (=170/3.8), thus greatly favoring aldehyde reduction. Formation of productive complexes, E.NAD(+) and E.NADH, leads to a 7- and 9-fold decrease of dissociation constants of initial binary complexes, respectively, demonstrating that 12-fold differential binding of NADH (K(i) = 16 microM) vs NAD(+) (K(i) = 195 microM) chiefly reflects difference in stabilities of E.NADH and E.NAD(+). Primary deuterium isotope effects on k(cat) and k(cat)/K(xylose) were, respectively, 1.55 +/- 0.09 and 2.09 +/- 0.31 in H(2)O, and 1.26 +/- 0.06 and 1.58 +/- 0.17 in D(2)O. No deuterium solvent isotope effect on k(cat)/K(xylose) was observed. When deuteration of coenzyme selectively slowed the hydride transfer step, (D)()2(O)(k(cat)/K(xylose)) was inverse (0.89 +/- 0.14). The isotope effect data suggest a chemical mechanism of carbonyl reduction by xylose reductase in which transfer of hydride ion is a partially rate-limiting step and precedes the proton-transfer step.     

The mechanism of direct heme transfer from the streptococcal cell surface protein Shp to HtsA of the HtsABC transporter

The heme-binding proteins Shp and HtsA are part of the heme acquisition machinery found in Streptococcus pyogenes. The hexacoordinate heme (Fe(II)-protoporphyrin IX) or hemochrome form of holoShp (hemoShp) is stable in air in Tris-HCl buffer, pH 8.0, binds to apoHtsA with a K(d) of 120 +/- 18 microm, and transfers its heme to apoHtsA with a rate constant of 28 +/- 6s(-1) at 25 degrees C, pH 8.0. The hemoHtsA product then autoxidizes to the hexacoordinate hemin (Fe(III)-protoporphyrin IX) or hemichrome form (hemiHtsA) with an apparent rate constant of 0.017 +/- 0.002 s(-1). HemiShp also rapidly transfers hemin to apoHtsA through a hemiShp.apoHtsA complex (K(d) = 48 +/- 7 microM) at a rate approximately 40,000 times greater than the rate of simple hemin dissociation from hemiShp into solvent (k(transfer) = 43 +/- 3s(-1) versus k(-hemin) = 0.0003 +/- 0.00006 s(-1)). The rate constants for hemin binding to and dissociation from HtsA (k'(hemin) approximately 80 microm(-1) s(-1), k(-hemin) = 0.0026 +/- 0.0002 s(-1)) are 50- and 10-fold greater than the corresponding rate constants for Shp (k(hemin) approximately 1.6 microM(-1) s(-1), k(-hemin) = 0.0003 s(-1)), which implies that HtsA has a more accessible active site. However, the affinity of apoHtsA for hemin (k(hemin) approximately 31,000 microm(-1)) is roughly 5-fold greater than that of apoShp (k(hemin) approximately 5,300 microM(-1)), accounting for the net transfer from Shp to HstA. These results support a direct, rapid, and affinity-driven mechanism of heme and hemin transfer from the cell surface receptor Shp to the ATP-binding cassette transporter system.     

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.     

Properties of the periplasmic nitrate reductases from Paracoccus pantotrophus and Escherichia coli after growth in tungsten-supplemented media

Paracoccus pantotrophus grown anaerobically under denitrifying conditions expressed similar levels of the periplasmic nitrate reductase (NAP) when cultured in molybdate- or tungstate-containing media. A native PAGE gel stained for nitrate reductase activity revealed that only NapA from molybdate-grown cells displayed readily detectable nitrate reductase activity. Further kinetic analysis showed that the periplasmic fraction from cells grown on molybdate (3 microM) reduced nitrate at a rate of V(max)=3.41+/-0.16 micromol [NO(3)(-)] min(-1) mg(-1) with an affinity for nitrate of K(m)=0.24+/-0.05 mM and was heat-stable up to 50 degrees C. In contrast, the periplasmic fraction obtained from cells cultured in media supplemented with tungstate (100 microM) reduced nitrate at a much slower rate, with much lower affinity (V(max)=0.05+/-0.002 micromol [NO(3)(-)] min(-1) mg(-1) and K(m)=3.91+/-0.45 mM) and was labile during prolonged incubation at >20 degrees C. Nitrate-dependent growth of Escherichia coli strains expressing only nitrate reductase A was inhibited by sub-mM concentrations of tungstate in the medium. In contrast, a strain expressing only NAP was only partially inhibited by 10 mM tungstate. However, none of the above experimental approaches revealed evidence that tungsten could replace molybdenum at the active site of E. coli NapA. The combined data show that tungsten can function at the active site of some, but not all, molybdoenzymes from mesophilic bacteria.     

Biophysical and kinetic characterization of HemAT, an aerotaxis receptor from Bacillus subtilis

HemAT from Bacillus subtilis is a new type of heme protein responsible for sensing oxygen. The structural and functional properties of the full-length HemAT protein, the sensor domain (1-178), and Tyr-70 mutants have been characterized. Kinetic and equilibrium measurements reveal that both full-length HemAT and the sensor domain show two distinct O(2) binding components. The high-affinity component has a K(dissociation) approximately 1-2 microM and a normal O(2) dissociation rate constant, k(O2) = 50-80 s(-1). The low-affinity component has a K(dissociation) approximately 50-100 microM and a large O(2) dissociation rate constant equal to approximately 2000 s(-1). The low n-value and biphasic character of the equilibrium curve indicate that O(2) binding to HemAT involves either independent binding to high- and low-affinity subunits in the dimer or negative cooperativity. Replacement of Tyr-70(B10) with Phe, Leu, or Trp in the sensor domain causes dramatic increases in k(O2) for both the high- and low-affinity components. In contrast, the rates and affinity for CO binding are little affected by loss of the Tyr-70 hydroxyl group. These results suggest highly dynamic behavior for the Tyr-70 side chain and the fraction of the "up" versus "down" conformation is strongly influenced by the nature of the iron-ligand complex. As a result of having both high- and low-affinity components, HemAT can respond to oxygen concentration gradients under both hypoxic (0-10 microM) and aerobic (50-250 microM) conditions, a property which could, in principle, be important for a robust sensing system. The unusual ligand-binding properties of HemAT suggest that asymmetry and apparent negative cooperativity play an important role in the signal transduction pathway.     

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.     

Reductive nitrosylation and peroxynitrite-mediated oxidation of heme-hemopexin

Hemopexin (HPX), which serves as a scavenger and transporter of toxic plasma heme, has been postulated to play a key role in the homeostasis of NO. In fact, HPX-heme(II) reversibly binds NO and facilitates NO scavenging by O(2). HPX-heme is formed by two four-bladed beta-propeller domains. The heme is bound between the two beta-propeller domains, residues His213 and His266 coordinate the heme iron atom. HPX-heme displays structural features of heme-proteins endowed with (pseudo-)enzymatic activities. In this study, the kinetics of rabbit HPX-heme(III) reductive nitrosylation and peroxynitrite-mediated oxidation of HPX-heme(II)-NO are reported. In the presence of excess NO, HPX-heme(III) is converted to HPX-heme(II)-NO by reductive nitrosylation. The second-order rate constant for HPX-heme(III) reductive nitrosylation is (1.3 +/- 0.1) x 10(1) m(-1).s(-1), at pH 7.0 and 10.0 degrees C. NO binding to HPX-heme(III) is rate limiting. In the absence and presence of CO2 (1.2 x 10(-3) m), excess peroxynitrite reacts with HPX-heme(II)-NO (2.6 x 10(-6) m) leading to HPX-heme(III) and NO, via the transient HPX-heme(III)-NO species. Values of the second-order rate constant for HPX-heme(III)-NO formation are (8.6 +/- 0.8) x 10(4) and (1.2 +/- 0.2) x 10(6) m(-1).s(-1) in the absence and presence of CO2, respectively, at pH 7.0 and 10.0 degrees C. The CO2-independent value of the first-order rate constant for HPX-heme(III)-NO denitrosylation is (4.3 +/- 0.4) x 10(-1) s(-1), at pH 7.0 and 10.0 degrees C. HPX-heme(III)-NO denitrosylation is rate limiting. HPX-heme(II)-NO appears to act as an efficient scavenger of peroxynitrite and of strong oxidants and nitrating species following the reaction of peroxynitrite with CO2 (e.g. ONOOC(O)O-, CO3-, and NO2).     

How does a valine residue that modulates heme-NO binding kinetics in inducible NO synthase regulate enzyme catalysis?

Nitric oxide (NO) release from nitric oxide synthases (NOSs) depends on the dissociation of a ferric heme-NO product complex (Fe^IIINO) that forms immediately after NO is made in the heme pocket. The NOS-like enzyme of Bacillus subtilis (bsNOS) has 10-20 fold slower Fe^IIINO dissociation rate (k_d) and NO association rate (k_on) compared to mammalian NOS counterparts. We previously showed that an Ile for Val substitution at the opening of the heme pocket in bsNOS contributes to these differences. The complementary mutation in mouse inducible NOS oxygenase domain (Val346Ile) decreased the NO k_on and k_d by 8 and 3-fold, respectively, compared to wild-type iNOSoxy, and also slowed the reductive processing of the heme-O₂ catalytic intermediate. To investigate how these changes affect steady-state catalytic behaviors, we generated and characterized the V346I mutant of full-length inducible NOS (iNOS). The mutant exhibited a 4-5 fold lower NO synthesis activity, an apparent uncoupled NADPH consumption, and formation of a heme-NO complex during catalysis that was no longer sensitive to solution NO scavenging. We found that these altered catalytic behaviors were not due to changes in the heme reduction rate or in the stability of the enzyme heme-O₂ intermediate, but instead were due to the slower NO k_on and k_d and a slower oxidation rate of the enzyme ferrous heme-NO complex. Computer simulations that utilized the measured kinetic values confirmed this interpretation, and revealed that the V346I iNOS has an enhanced NADPH-dependent NO dioxygenase activity that converts almost 1 NO to nitrate for every NO that the enzyme releases into solution. Together, our results highlight the importance of heme pocket geometry in tuning the NO release versus NO dioxygenase activities of iNOS.     

Comparative kinetics of cofactor association and dissociation for the human and trypanosomal S-adenosylhomocysteine hydrolases. 1. Basic features of the association and dissociation processes

The S-adenosyl-l-homocysteine (AdoHcy) hydrolases catalyze the reversible conversion of AdoHcy to adenosine and homocysteine, making use of a catalytic cycle in which a tightly bound NAD+ oxidizes the 3-hydroxyl group of the substrate at the beginning of the cycle, activating the 4-CH bond for elimination of homocysteine, followed by Michael addition of water to the resulting intermediate and a final reduction by the tightly bound NADH to give adenosine. The equilibrium and kinetic properties of the association and dissociation of the cofactor NAD+ from the enzymes of Homo sapiens (Hs-SAHH) and Trypanosoma cruzi (Tc-SAHH) are qualitatively similar but quantitatively distinct. Both enzymes bind NAD+ in a complex scheme. The four active sites of the homotetrameric apoenzyme appear to divide into two numerically equal classes of active sites. One class of sites binds cofactor weakly and generates full activity very rapidly (in less than 1 min). The other class binds cofactor more strongly but generates activity only slowly (>30 min). In the case of Tc-SAHH, the final affinity for NAD+ is roughly micromolar and this affinity persists as the equilibrium affinity. In the case of Hs-SAHH, the slow-binding phase terminates in micromolar affinity also, but over a period of hours, the dissociation rate constant decreases until the final equilibrium affinity is in the nanomolar range. The slow binding of NAD+ by both enzymes exhibits saturation kinetics with respect to the cofactor concentration; however, binding to Hs-SAHH has a maximum rate constant around 0.06 s-1, while the rate constant for binding to Tc-SAHH levels out at 0.006 s-1. In contrast to the complex kinetics of association, both enzymes undergo dissociation of NAD+ from all four sites in a single first-order reaction. The equilibrium affinities of both Hs-SAHH and Tc-SAHH for NADH are in the nanomolar range. The dissociation rate constants and the slow-binding association rate constants for NAD+ show a complex temperature dependence with both enzymes; however, the cofactor always dissociates more rapidly from Tc-SAHH than from Hs-SAHH, the ratio being around 80-fold at 37 degrees C, and the cofactor binds more rapidly to Hs-SAHH than to Tc-SAHH above approximately 16 degrees C. These features present an opening for selective inhibition of Tc-SAHH over Hs-SAHH, demonstrated with the thioamide analogues of NAD+ and NADH. Both analogues bind to Hs-SAHH with approximately 40 nM affinities but much more weakly to Tc-SAHH (0.6-15 microM). Nevertheless, both analogues inactivated Tc-SAHH 60% (NAD+ analogue) or 100% (NADH analogue) within 30 min, while the degree of inhibition of Hs-SAHH approached 30% only after 12 h. The rate of loss of activity is equal to the rate of dissociation of the cofactor and thus 80-fold faster at 37 degrees C for Tc-SAHH.     

Nitric oxide metabolism in mammalian cells: substrate and inhibitor profiles of a NADPH-cytochrome P450 oxidoreductase-coupled microsomal nitric oxide dioxygenase

Human intestinal Caco-2 cells metabolize and detoxify NO via a dioxygen- and NADPH-dependent, cyanide- and CO-sensitive pathway that yields nitrate. Enzymes catalyzing NO dioxygenation fractionate with membranes and are enriched in microsomes. Microsomal NO metabolism shows apparent KM values for NO, O2, and NADPH of 0.3, 9, and 2 microM, respectively, values similar to those determined for intact or digitonin-permeabilized cells. Similar to cellular NO metabolism, microsomal NO metabolism is superoxide-independent and sensitive to heme-enzyme inhibitors including CO, cyanide, imidazoles, quercetin, and allicin-enriched garlic extract. Selective inhibitors of several cytochrome P450s and heme oxygenase fail to inhibit the activity, indicating limited roles for a subset of microsomal heme enzymes in NO metabolism. Diphenyleneiodonium and cytochrome c(III) inhibit NO metabolism, suggesting a role for the NADPH-cytochrome P450 oxidoreductase (CYPOR). Involvement of CYPOR is demonstrated by the specific inhibition of the NO metabolic activity by inhibitory anti-CYPOR IgG. In toto, the results suggest roles for a microsomal CYPOR-coupled and heme-dependent NO dioxygenase in NO metabolism, detoxification, and signal attenuation in mammalian cells.     

*NO dissociation represents the rate limiting step for O2-mediated oxidation of ferrous nitrosylated Mycobacterium leprae truncated hemoglobin O

Mycobacterium leprae truncated hemoglobin O (trHbO) protects from nitrosative stress and sustains mycobacterial respiration. Here, kinetics of M. leprae trHbO(II)-NO denitrosylation and of O(2)-mediated oxidation of M. leprae trHbO(II)-NO are reported. Values of the first-order rate constant for *NO dissociation from M. leprae trHbO(II)-NO (k(off)) and of the first-order rate constant for O(2)-mediated oxidation of M. leprae trHbO(II)-NO (h) are 1.3 x 10(-4) s(-1) and 1.2 x 10(-4) s(-1), respectively. The coincidence of values of k(off) and h suggests that O(2)-mediated oxidation of M. leprae trHbO(II)-NO occurs with a reaction mechanism in which *NO, that is initially bound to heme(II), is displaced by O(2) but may stay trapped in a protein cavity(ies) close to heme(II). Next, M. leprae trHbO(II)-O(2) reacts with *NO giving the transient Fe(III)-OONO species preceding the formation of the final product M. leprae trHbO(III). *NO dissociation from heme(II)-NO represents the rate limiting step for O(2)-mediated oxidation of M. leprae trHbO(II)-NO.     

Nitric oxide reacts with the single-electron reduced active site of cytochrome c oxidase

The reduction kinetics of the mutants K354M and D124N of the Paracoccus denitrificans cytochrome oxidase (heme aa(3)) by ruthenium hexamine was investigated by stopped-flow spectrophotometry in the absence/presence of NO. Quick heme a reduction precedes the biphasic heme a(3) reduction, which is extremely slow in the K354M mutant (k(1) = 0.09 +/- 0.01 s(-1); k(2) = 0.005 +/- 0.001 s(-1)) but much faster in the D124N aa(3) (k(1) = 21 +/- 6 s(-1); k(2) = 2.2 +/- 0.5 s(-1)). NO causes a very large increase (>100-fold) in the rate constant of heme a(3) reduction in the K354M mutant but only a approximately 5-fold increase in the D124N mutant. The K354M enzyme reacts rapidly with O(2) when fully reduced but is essentially inactive in turnover; thus, it was proposed that impaired reduction of the active site is the cause of activity loss. Since at saturating [NO], heme a(3) reduction is approximately 100-fold faster than the extremely low turnover rate, we conclude that, contrary to O(2), NO can react not only with the two-electron but also with the single-electron reduced active site. This mechanism would account for the efficient inhibition of cytochrome oxidase activity by NO in the wild-type enzyme, both from P. denitrificans and from beef heart. Results also suggest that the H(+)-conducting K pathway, but not the D pathway, controls the kinetics of the single-electron reduction of the active site.     

Human aldehyde dehydrogenase: coenzyme binding studies

The binding of NADH and NAD+ to the human liver cytoplasmic, E1, and mitochondrial, E2, isozymes at pH 7.0 and 25 degrees C was studied by the NADH fluorescence enhancement technique, the sedimentation technique, and steady-state kinetics. The binding of radiolabeled [14C]NADH and [14C]NAD+ to the E1 isozyme when measured by the sedimentation technique yielded linear Scatchard plots with a dissociation constant of 17.6 microM for NADH and 21.4 microM for NAD+ and a stoichiometry of ca. two coenzyme molecules bound per enzyme tetramer. The dissociation constant, 19.2 microM, for NADH as competitive inhibitor was found from steady-state kinetics. With the mitochondrial E2 isozyme, the NADH fluorescence enhancement technique showed only one, high-affinity binding site (KD = 0.5 microM). When the sedimentation technique and radiolabeled coenzymes were used, the binding studies showed nonlinear Scatchard plots. A minimum of two binding sites with lower affinity was indicated for NADH (KD = 3-6 microM and KD = 25-30 microM) and also for NAD+ (KD = 5-7 microM and KD = 15-30 microM). A fourth binding site with the lowest affinity (KD = 184 microM for NADH and KD = 102 microM for NAD+) was observed from the steady-state kinetics. The dissociation constant for NAD+, determined by the competition with NADH via fluorescence titration, was found to be 116 microM. The number of binding sites found by the fluorescence titration (n = 1 for NADH) differs from that found by the sedimentation technique (n = 1.8-2.2 for NADH and n = 1.2-1.6 for NAD+).(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphodiesterase A1, a regulator of cellulose synthesis in Acetobacter xylinum, is a heme-based sensor

The phosphodiesterase A1 protein of Acetobacter xylinum, AxPDEA1, is a key regulator of bacterial cellulose synthesis. This phosphodiesterase linearizes cyclic bis(3'-->5')diguanylic acid, an allosteric activator of the bacterial cellulose synthase, to the ineffectual pGpG. Here we show that AxPDEA1 contains heme and is regulated by reversible binding of O(2) to the heme. Apo-AxPDEA1 has less than 2% of the phosphodiesterase activity of holo-AxPDEA1, and reconstitution with hemin restores full activity. O(2) regulation is due to deoxyheme being a better activator than oxyheme. AxPDEA1 is homologous to the Escherichia coli direct oxygen sensor protein, EcDos, over its entire length and is homologous to the FixL histidine kinases over only a heme-binding PAS domain. The properties of the heme-binding domain of AxPDEA1 are significantly different from those of other O(2)-responsive heme-based sensors. The rate of AxPDEA1 autoxidation (half-life > 12 h) is the slowest observed so far for this type of heme protein fold. The O(2) affinity of AxPDEA1 (K(d) approximately 10 microM) is comparable to that of EcDos, but the rate constants for O(2) association (k(on) = 6.6 microM(-)(1) s(-)(1)) and dissociation (k(off) = 77 s(-)(1)) are 2000 times higher. Our results illustrate the versatility of signal transduction mechanisms for the heme-PAS class of O(2) sensors and provide the first example of O(2) regulation of a second messenger.     

Flavohemoglobin detoxifies nitric oxide in aerobic, but not anaerobic, Escherichia coli. Evidence for a novel inducible anaerobic nitric oxide-scavenging activity.

Nitric-oxide dioxygenase (NOD) and reductase (NOR) activities of flavohemoglobin (flavoHb) have been suggested as mechanisms for NO metabolism and detoxification in a variety of microbes. Mechanisms of NO detoxification were tested in Escherichia coli using flavoHb-deficient mutants and overexpressors. flavoHb showed negligible anaerobic NOR activity and afforded no protection to the NO-sensitive aconitase or the growth of anoxic E. coli, whereas the NOD activity and the protection afforded with O(2) were substantial. A NO-inducible, O(2)-sensitive, and cyanide-resistant NOR activity efficiently metabolized NO and protected anaerobic cells from NO toxicity independent of the NOR activity of flavoHb. flavoHb possesses nitrosoglutathione and nitrite reductase activities that may account for the protection it affords against these agents. NO detoxification by flavoHb occurs most effectively via O(2)-dependent NO dioxygenation.     

Chemical and steady-state kinetic analyses of a heterologously expressed heme dependent chlorite dismutase

Chlorite dismutase carries out the heme-catalyzed decomposition of ClO2- to Cl- and O2, an unusual transformation with biotechnological and bioremediative applications. The enzyme has been successfully overexpressed for the first time in highly functional form in Escherichia coli and its steady state kinetics studied. The purified enzyme is abundant (55 mg/L cell culture), highly active (approximately 4.7 x 10(3) micromol of ClO2- min(-1) mg(-1) subunit) and nearly stoichiometric in heme; further, it shares spectroscopic and physicochemical features with chlorite dismutases previously isolated from three organisms. A careful study of the enzyme's steady state kinetics has been carried out. ClO2- consumption and O2 release rates were measured, yielding comparable values of kcat (4.5 x 10(5) min(-1)), K(m) (approximately 215 microM), and kcat/Km (3.5 x 10(7) M(-1) s(-1) via either method (4 degrees C, pH 6.8; all values referenced per heme-containing subunit). ClO2-:O2 stoichiometry exhibited a 1:1 relationship under all conditions measured. Though the value of kcat/Km indicates near diffusion control of the reaction, viscosogens had no effect on k(cat)/K(m) or V(max). The product O2 did not inhibit the reaction at saturating [O2], but Cl- is a mixed inhibitor with relatively high values of KI (225 mM for enzyme and 95.6 mM for the enzyme-substrate complex), indicating a relatively low affinity of the heme iron for halogen ions. Chlorite irreversibly inactivates the enzyme after approximately 1.7 x 10(4) turnovers (per heme) and with a half-life of 0.39 min, resulting in bleaching of the heme chromophore. The inactivation K(I) (K(inact)) of 166 microM is similar in magnitude to Km, consistent with a common Michaelis complex on the pathway to both reaction and inactivation. The one-electron peroxidase substrate guaiacol offers incomplete protection of the enzyme from inactivation. Mechanisms in keeping with the available data and the properties of other well-described heme enzymes are proposed.     

Role of the low-affinity binding site in electron transfer from cytochrome C to cytochrome C peroxidase

The interaction of yeast iso-1-cytochrome c (yCc) with the high- and low-affinity binding sites on cytochrome c peroxidase compound I (CMPI) was studied by stopped-flow spectroscopy. When 3 microM reduced yCc(II) was mixed with 0.5 microM CMPI at 10 mM ionic strength, the Trp-191 radical cation was reduced from the high-affinity site with an apparent rate constant >3000 s(-1), followed by slow reduction of the oxyferryl heme with a rate constant of only 10 s(-1). In contrast, mixing 3 microM reduced yCc(II) with 0.5 microM preformed CMPI *yCc(III) complex led to reduction of the radical cation with a rate constant of 10 s(-1), followed by reduction of the oxyferryl heme in compound II with the same rate constant. The rate constants for reduction of the radical cation and the oxyferryl heme both increased with increasing concentrations of yCc(II) and remained equal to each other. These results are consistent with a mechanism in which both the Trp-191 radical cation and the oxyferryl heme are reduced by yCc(II) in the high-affinity binding site, and the reaction is rate-limited by product dissociation of yCc(III) from the high-affinity site with apparent rate constant k(d). Binding yCc(II) to the low-affinity site is proposed to increase the rate constant for dissociation of yCc(III) from the high-affinity site in a substrate-assisted product dissociation mechanism. The value of k(d) is <5 s(-1) for the 1:1 complex and >2000 s(-1) for the 2:1 complex at 10 mM ionic strength. The reaction of horse Cc(II) with CMPI was greatly inhibited by binding 1 equiv of yCc(III) to the high-affinity site, providing evidence that reduction of the oxyferryl heme involves electron transfer from the high-affinity binding site rather than the low-affinity site. The effects of CcP surface mutations on the dissociation rate constant indicate that the high-affinity binding site used for the reaction in solution is the same as the one identified in the yCc*CcP crystal structure.     

Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.