The nitric oxide synthases (NOS) are the only heme-containing enzymes that require tetrahydrobiopterin (BH4) as a cofactor. Previous studies indicate that only the fully reduced (i.e., tetrahydro) form of BH4 can support NO synthesis. Here, we characterize pterin-free inducible NOS (iNOS) and iNOS reconstituted with eight different tetrahydro- or dihydropterins to elucidate how changes in pterin side-chain structure and ring oxidation state regulate iNOS. Seven different enzyme properties that are important for catalysis and are thought to involve pterin were studied. Only two properties were found to depend on pterin oxidation state (i.e., they required fully reduced tetrahydropterins) and were independent of side chain structure: NO synthesis and the ability to increase heme-dependent NADPH oxidation in response to substrates. In contrast, five properties were exclusively dependent on pterin side-chain structure or stereochemistry and were independent of pterin oxidation state: pterin binding affinity, and its ability to shift the heme iron to its high-spin state, stabilize the ferrous heme iron coordination structure, support heme iron reduction, and promote iNOS subunit assembly into a dimer. These results clarify how structural versus redox properties of the pterin impact on its multifaceted role in iNOS function. In addition, the data reveal that during NO synthesis all pterin-dependent steps up to and including heme iron reduction can take place independent of the pterin ring oxidation state, indicating that the requirement for fully reduced pterin occurs at a point in catalysis beyond heme iron reduction.
We conclude that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself.
We studied steps that make up the initial and steadystate phases of nitric oxide (NO) synthesis to understand how activity of bovine endothelial NO synthase (eNOS) is regulated. Stopped-flow analysis of NADPH-dependent flavin reduction showed the rate increased from 0.13 to 86 s ؊1 upon calmodulin binding, but this supported slow heme reduction in the presence of either Arg or N -hydroxy-L-arginine (0.005 and 0.014 s ؊1 , respectively, at 10°C). O 2 binding to ferrous eNOS generated a transient ferrous dioxy species (Soret peak at 427 nm) whose formation and decay kinetics indicate it can participate in NO synthesis. The kinetics of heme-NO complex formation were characterized under anaerobic conditions and during the initial phase of NO synthesis. During catalysis heme-NO complex formation required buildup of relatively high solution NO concentrations (>50 nM), which were easily achieved with N -hydroxy-L-arginine but not with Arg as substrate. Heme-NO complex formation caused eNOS NADPH oxidation and citrulline synthesis to decrease 3-fold and the apparent K m for O 2 to increase 6-fold. Our main conclusions are: 1) The slow steady-state rate of NO synthesis by eNOS is primarily because of slow electron transfer from its reductase domain to the heme, rather than heme-NO complex formation or other aspects of catalysis. 2) eNOS forms relatively little heme-NO complex during NO synthesis from Arg, implying NO feedback inhibition has a minimal role. These properties distinguish eNOS from the other NOS isoforms and provide a foundation to better understand its role in physiology and pathology. Nitric-oxide synthases (NOSs)1 catalyze a stepwise oxidation of L-arginine (Arg) to citrulline and nitric oxide (NO) (1-3). In mammals, three NOSs are expressed that differ in their primary sequence, post-translational modifications, cellular location, and tissue expression (4 -6), consistent with their participating in a range of physiologic and pathologic systems. Two NOSs (neuronal, nNOS or NOS-I; and endothelial, eNOS or NOS-III) are constitutively expressed and participate in signal cascades by synthesizing NO in response to Ca 2ϩ -dependent CaM binding. A third NOS (cytokine-inducible, iNOS or NOS-II) is primarily regulated by transcriptional mechanisms, binds CaM irrespective of the Ca 2ϩ concentration to be always active, and functions as both a regulator and effector of the immune response.Although NO synthesis activities of the NOS isoforms differ considerably, how and why this occurs is unclear. A comparison of published steady-state rates shows that eNOS is about four to eight times slower than either nNOS or iNOS (7-14). Because NO synthesis is actually the result of many steps, it is imperative to identify which steps limit the activity of a particular NOS isoform. Work with NOS chimeras containing swapped reductase domains has suggested heme reduction could be responsible for the low activity of eNOS (30). However, it seems that NOS catalysis is comprised of two parts (15, 16): an active component that includes...
The nitric oxide synthases (NOS) are heme-containing enzymes responsible for catalyzing the fiveelectron oxidation of a guanidino nitrogen of L-arginine to produce the free radical nitric oxide. The binding sites of the heme group, as well as of the L-arginine substrate and tetrahydrobiopterin cofactor, are located within the oxygenase domain of the NOS enzymes. Reduction of the heme is the first committed step in catalysis, as this allows for binding and activation of molecular oxygen, followed by oxidative attack on the L-arginine substrate. As with heme groups in other enzymes, the electronic properties of the NOS heme are modified by substrate and cofactor binding in its vicinity. Here we present the first quantitative thermodynamic data of the NOS heme with the determination of the heme midpoint reduction potentials for the neuronal NOS and inducible NOS oxygenase domains. In the absence of L-arginine and tetrahydrobiopterin, the midpoint potential of the inducible NOS oxygenase heme iron is over 100 mV lower than that of the neuronal NOS oxygenase heme iron. Binding of the substrate alone, cofactor alone, or both combined with the inducible NOS oxygenase increases the heme iron reduction potential by 112, 52, and 84 mV, respectively. On the basis of these data, we calculate that the binding affinities of L-arginine and tetrahydrobiopterin increase by about 80-fold and 8-fold, respectively, for the reduced heme iron form of the enzyme. These data support interactive binding of L-arginine and tetrahydrobiopterin in proximity to the inducible NOS heme group, as observed in the crystal structure of this enzyme. In contrast, addition of L-arginine, tetrahydrobiopterin, or both to neuronal NOS oxygenase do not markedly change its heme iron midpoint potential, with observed shifts of +19, -18, and -10 mV, respectively. These data explain the contrasting reactivities between the two NOS isoforms regarding their different NADPH consumption rates and capacity to support heme iron reduction and are indicative of the regulatory mechanisms that each enzyme employs toward electron transfer. We also examine the effects of three substrate-based inhibitors of NOS on the heme iron midpoint potentials. Among these inhibitors, S-ethylisothiourea decreased the heme potentials of tetrahydrobiopterin-bound inducible NOS and neuronal NOS by 27 and 24 mV, respectively, N-nitro-L-arginine methyl ester lowered both potentials below -460 mV, and aminoguanidine slightly increased both potentials. This work suggests the following:(1) A thermodynamic block of reductase-catalyzed heme reduction exists in inducible NOS but not in neuronal NOS in the absence of substrate and tetrahydrobiopterin. This reveals distinct heme environments for the two isoforms. (2) Heme iron reduction thermodynamics in inducible NOS are improved by tetrahydrobiopterin and L-arginine, implying that this isoform is uniquely configured to respond to substrate and pterin control.(3) Some, but not all, inhibitors that reduce electron flux through NOS act by affecting...
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