Chin Chuan Wei

Rapid Kinetic Studies Link Tetrahydrobiopterin Radical Formation to Heme-dioxy Reduction and Arginine Hydroxylation in Inducible Nitric-oxide Synthase

To understand how heme and (6R)-5,6,7,8-tetrahydro-l-biopterin (H4B) participate in nitric-oxide synthesis, we followed ferrous-dioxy heme (FeIIO2) formation and disappearance, H4B radical formation, and Arg hydroxylation during a single catalytic turnover by the inducible nitric-oxide synthase oxygenase domain (iNOSoxy). In all cases, prereduced (ferrous) enzyme was rapidly mixed with an O2-containing buffer to start the reaction. A ferrous-dioxy intermediate formed quickly (53 s−1) and then decayed with concurrent buildup of ferric iNOSoxy. The buildup of the ferrous-dioxy intermediate preceded both H4B radical formation and Arg hydroxylation. However, the rate of ferrous-dioxy decay (12 s−1) was equivalent to the rate of H4B radical formation (11 s−1) and the rate of Arg hydroxylation (9 s−1). Practically all bound H4B was oxidized to a radical during the reaction and was associated with hydroxylation of 0.6 mol of Arg/mol of heme. In dihydrobiopterin-containing iNOSoxy, ferrous-dioxy decay was much slower and was not associated with Arg hydroxylation. These results establish kinetic and quantitative links among ferrous-dioxy disappearance, H4B oxidation, and Arg hydroxylation and suggest a mechanism whereby H4B transfers an electron to the ferrous-dioxy intermediate to enable the formation of a heme-based oxidant that rapidly hydroxylates Arg.

Catalytic Reduction of a Tetrahydrobiopterin Radical within Nitric-oxide Synthase

Nitric-oxide synthases (NOS) are catalytically self-sufficient flavo-heme enzymes that generate NO from arginine (Arg) and display a novel utilization of their tetrahydrobiopterin (H4B) cofactor. During Arg hydroxylation, H4B acts as a one-electron donor and is then presumed to redox cycle (i.e. be reduced back to H4B) within NOS before further catalysis can proceed. Whereas H4B radical formation is well characterized, the subsequent presumed radical reduction has not been demonstrated, and its potential mechanisms are unknown. We investigated radical reduction during a single turnover Arg hydroxylation reaction catalyzed by neuronal NOS to document the process, determine its kinetics, and test for involvement of the NOS flavoprotein domain. We utilized a freeze-quench instrument, the biopterin analog 5-methyl-H4B, and a method that could separately quantify the flavin and pterin radicals that formed in NOS during the reaction. Our results establish that the NOS flavoprotein domain catalyzes reduction of the biopterin radical following Arg hydroxylation. The reduction is calmodulin-dependent and occurs at a rate that is similar to heme reduction and fast enough to explain H4B redox cycling in NOS. These results, in light of existing NOS crystal structures, suggest a “through-heme” mechanism may operate for H4B radical reduction in NOS.

Bacterial Flavodoxins Support Nitric Oxide Production by Bacillus subtilis Nitric-oxide Synthase

Unlike animal nitric-oxide synthases (NOSs), the bacterial NOS enzymes have no attached flavoprotein domain to reduce their heme and so must rely on unknown bacterial proteins for electrons. We tested the ability of two Bacillus subtilis flavodoxins (YkuN and YkuP) to support catalysis by purified B. subtilis NOS (bsNOS). When an NADPH-utilizing bacterial flavodoxin reductase (FLDR) was added to reduce YkuP or YkuN, both supported NO synthesis from either l-arginine or N-hydroxyarginine and supported a linear nitrite accumulation over a 30-min reaction period. Rates of nitrite production were directly dependent on the ratio of YkuN or YkuP to bsNOS. However, the V/Km value for YkuN (5.2 × 105) was about 20 times greater than that of YkuP (2.6 × 104), indicating YkuN is more efficient in supporting bsNOS catalysis. YkuN that was either photo-reduced or prereduced by FLDR transferred an electron to the bsNOS ferric heme at rates similar to those measured for heme reduction in the animal NOSs. YkuN supported a similar NO synthesis activity by a different bacterial NOS (Deinococcus radiodurans) but not by any of the three mammalian NOS oxygenase domains nor by an insect NOS oxygenase domain. Our results establish YkuN as a kinetically competent redox partner for bsNOS and suggest that FLDR/flavodoxin proteins could function physiologically to support catalysis by bacterial NOSs.

Why do nitric oxide synthases use tetrahydrobiopterin

We are combining stopped-flow, stop-quench, and rapid-freezing kinetic methods to help clarify the unique redox roles of tetrahydrobiopterin (H(4)B) in NO synthesis, which occurs via the consecutive oxidation of L-arginine (Arg) and N-hydroxy-L-arginine (NOHA). In the Arg reaction, H(4)B radical formation is coupled to reduction of a heme Fe(II)O(2) intermediate. The tempo of this electron transfer is important for coupling Fe(II)O(2) formation to Arg hydroxylation. Because H(4)B provides this electron faster than can the NOS reductase domain, H(4)B appears to be a kinetically preferred source of the second electron for oxygen activation during Arg hydroxylation. A conserved Trp (W457 in mouse inducible NOS) has been shown to influence product formation by controlling the kinetics of H(4)B electron transfer to the Fe(II)O(2) intermediate. This shows that the NOS protein tunes H(4)B redox function. In the NOHA reaction the role of H(4)B is more obscure. However, existing evidence suggests that H(4)B may perform consecutive electron donor and acceptor functions to reduce the Fe(II)O(2) intermediate and then ensure that NO is produced from NOHA.

A Conserved Tryptophan 457 Modulates the Kinetics and Extent of N-Hydroxy-l-Arginine Oxidation by Inducible Nitric-oxide Synthase

In the oxygenase domain of mouse inducible nitric-oxide synthase (iNOSoxy), a conserved tryptophan residue, Trp-457, regulates the kinetics and extent of l-Arg oxidation toN ω-hydroxy-l-arginine (NOHA) by controlling electron transfer between bound (6R)-tetrahydrobiopterin (H4B) cofactor and the enzyme heme FeIIO2 intermediate (Wang, Z. Q., Wei, C. C., Ghosh, S., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) Biochemistry 40, 12819–12825). To investigate whether NOHA oxidation to citrulline and nitric oxide (NO) is regulated by a similar mechanism, we performed single turnover reactions with wild type iNOSoxy and mutants W457F and W457A. Ferrous proteins containing NOHA plus H4B or NOHA plus 7,8-dihydrobiopterin (H2B), were mixed with O2-containing buffer, and then heme spectral transitions and product formation were followed versus time. All three proteins formed a FeIIO2 intermediate with identical spectral characteristics. In wild type, H4B increased the disappearance rate of the FeIIO2intermediate relative to H2B, and its disappearance was coupled to the formation of a FeIIINO immediate product prior to formation of ferric enzyme. In W457F and W457A, the disappearance rate of the FeIIO2 intermediate was slower than in wild type and took place without detectable build-up of the heme FeIIINO immediate product. Rates of FeIIO2 disappearance correlated with rates of citrulline formation in all three proteins, and reactions containing H4B formed 1.0, 0.54, and 0.38 citrulline/heme in wild type, W457F, and W457A iNOSoxy, respectively. Thus, Trp-457 modulates the kinetics of NOHA oxidation by iNOSoxy, and this is important for determining the extent of citrulline and NO formation. Our findings support a redox role for H4B during NOHA oxidation to NO by iNOSoxy.

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(III)NO) 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(III)NO 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-O(2) 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-O(2) 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.

Lys842 in neuronal nitric-oxide synthase enables the autoinhibitory insert to antagonize calmodulin binding, increase FMN shielding, and suppress interflavin electron transfer.

Neuronal nitric-oxide synthase (nNOS) contains a unique autoinhibitory insert (AI) in its FMN subdomain that represses nNOS reductase activities and controls the calcium sensitivity of calmodulin (CaM) binding to nNOS. How the AI does this is unclear. A conserved charged residue (Lys842) lies within a putative CaM binding helix in the middle of the AI. We investigated its role by substituting residues that neutralize (Ala) or reverse (Glu) the charge at Lys842. Compared with wild type nNOS, the mutant enzymes had greater cytochrome c reductase and NADPH oxidase activities in the CaM-free state, were able to bind CaM at lower calcium concentration, and had lower rates of heme reduction and NO synthesis in one case (K842A). Moreover, stopped-flow spectrophotometric experiments with the nNOS reductase domain indicate that the CaM-free mutants had faster flavin reduction kinetics and had less shielding of their FMN subdomains compared with wild type and no longer increased their level of FMN shielding in response to NADPH binding. Thus, Lys842 is critical for the known functions of the AI and also enables two additional functions of the AI as newly identified here: suppression of electron transfer to FMN and control of the conformational equilibrium of the nNOS reductase domain. Its effect on the conformational equilibrium probably explains suppression of catalysis by the AI.

Effective adsorption of phenols using nitrogen-containing porous activated carbon prepared from sunflower plates

Nitrogen-containing porous carbons, the 800SP-NH3, were synthesized using sunflower plates as the major carbon source carbonized at 800°C and activated with concentrated aqueous ammonia at the same temperature. The porous carbons were characterized by nitrogen physical adsorption-desorption, surface area analyzer, FT-IR, and SEM. The adsorption properties of the porous carbons towards phenols were also investigated by batch methods. The test results show that the average pore diameter of porous carbon is smaller than 2 nm, and nitrogen-containing chemical groups are formed on its surface. The adsorption capacity for phenol, 4-chlorophenol, and p-nitrophenol is 316.5mg/g, 330.24mg/g and 387.62mg/g due to its developed pore structure and nitrogen-containing chemical groups. The adsorption isotherm data greatly obey the Langmuir model.

Conformational States and Kinetics of the Calcium Binding Domain of NADPH Oxidase 5

Superoxide generated by human NADPH oxidase 5 (NOX5) is of growing importance for various physiological and pathological processes. The activity of NOX5 appears to be regulated by a self-contained Ca2+ binding domain (CaBD). Recently Bánfi et al. suggest that the conformational change of CaBD upon Ca2+ binding is essential for domain-domain interaction and superoxide production. The authors studied its structural change using intrinsic Trp fluorescence and hydrophobic dye binding; however, their conformational study was not thorough and the kinetics of metal binding was not demonstrated. Here we generated the recombinant CaBD and an E99Q/E143Q mutant to characterize them using fluorescence spectroscopy. Ca2+ binding to CaBD induces a conformational change that exposes hydrophobic patches and increases the quenching accessibilities of its Trp residues and AEDANS at Cys107. The circular dichroism spectra indicated no significant changes in the secondary structures of CaBD upon metal binding. Stopped-flow spectrometry revealed a fast Ca2+ dissociation from the N-terminal half, followed by a slow Ca2+ dissociation from the C-terminal half. Combined with a chemical stability study, we concluded that the C-terminal half of CaBD has a higher Ca2+ binding affinity, a higher chemical stability, and a slow Ca2+ dissociation. The Mg2+-bound CaBD was also investigated and the results indicate that its structure is similar to the apo form. The rate of Mg2+ dissociation was close to that of Ca2+ dissociation. Our data suggest that the N- and C-terminal halves of CaBD are not completely structurally independent.

Nitric oxide synthase: use of stopped-flow spectroscopy and rapid-quench methods in single-turnover conditions to examine formation and reactions of heme-O2 intermediate in early catalysis.

The chapter describes the study of nitric oxide synthases (NOS) catalysis in a single turnover setting using stopped-flow and stop-quench techniques. More general reviews of these methods and of NOS biochemistry are available. Stopped-flow spectroscopy is useful for investigating reaction kinetics of the NOS Fe II O 2 intermediate. This method may be combined with other techniques—such as rapid chemical quenching and rapid freezing—to investigate temporal and quantitative links between Fe II O 2 formation and disappearance, L-arginine (Arg) or N ω -hydroxy-L-arginine (NOHA) oxidation, and 6R-tetrahydrobiopterin (H 4 B) radical formation in NOS. Application of these three methods within a single-turnover setting is further defining the NOS reaction mechanism. Several aspects of NOS make rapid-mixing single-turnover studies particularly well suited to investigating the reaction mechanism: the heme binds and activates O2 twice in two experimentally separable catalytic cycles to generate NO from Arg. A future challenge will be to create reaction conditions or NOS mutant proteins that stabilize heme intermediates other than the Fe II O2 species, such that they build to detectable levels during the NOS single-turnover reaction.