L-arginine and calmodulin regulation of the heme iron reactivity in neuronal nitric oxide synthase.

Matsuoka, Ariki; Stuehr, Dennis J.; Olson, John S.; Clark, Pamela; Ikeda‐Saito, Masao

doi:10.1016/s0021-9258(17)31996-8

Cited by 117 publications

(54 citation statements)

References 34 publications

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“…The current results offer new insights in catalysis by the heme of NO synthases. First, we extend prior studies (Wang et al, 1994;Matsuoka et al, 1994) by demonstrating the close proximity of the binding site of L-arginine analogues and inhibitors to the heme catalytic active site. While interactions between substrates and heme-bound ligand have been predicted by several catalytic models (Feldman et al, 1993;Marletta, 1993), the data we report here are consistent with those models, though more direct evidence is required to firmly establish the exact location of L-arginine and BH 4 in the distal pocket.…”

Section: Discussionsupporting

confidence: 56%

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Interactions between Substrate Analogues and Heme Ligands in Nitric Oxide Synthase

1997

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The substrate binding site in nitric oxide synthase (NOS) can accommodate the physiological substrates, L-arginine and N ω -hydroxy L-arginine as well as many substrate analogues and inhibitors. Resonance Raman spectra of carbon monoxide-bound NOS were measured to determine how these substrates and analogues interact with heme, the prosthetic group which activates oxygen for the catalytic generation of NO and citrulline from arginine in the enzyme. Two distinct conformations of the Fe-C-O moiety were detected in the resonance Raman spectra, although in the optical absorption spectra the two species are indistinguishable. In one, termed the β-form, the Fe-CO stretching frequency and the C-O stretching frequency, located at ∼487 and ∼1949 cm -1 , respectively, demonstrate that the Fe-C-O group adopts a linear conformation perpendicular to the heme plane ("open" structure). In the other, termed the R-form, frequencies of ∼502 and ∼1929 cm -1 , respectively, indicate that the binding properties of bound CO are significantly affected by its immediate environment thereby leading to a "closed" structure. In the presence of L-arginine or N ω -OH-L-arginine all of the molecules exhibit the closed structure, indicating that the substrates exert a strong polar (and/or steric) effect on the hemebound ligand. In the absence of any substrate or inhibitor only half of the heme population adopts the open structure whereas the rest of the heme content retains the closed conformation. It is proposed that in this population with the closed structure tetrahydrobiopterin, a cofactor of NO synthase, may reside in close proximity to the heme-bound ligand and interact with it in a similar manner as do substrates. The inverse correlation between the Fe-CO and C-O stretching modes suggests that in NOS the bonding of the cysteine to the heme iron may be weaker, as found in chloroperoxidase, than in cytochrome P-450 enzymes. This work continually proves resonance Raman spectroscopy as a powerful probe for the interactions between substrate/inhibitor and the heme active site of proteins.

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Section: Discussionsupporting

confidence: 56%

“…It appears that the substrate binding site is occupied by some group in half of the population. This conclusion is supported by the CO binding rate constants determined by Matsuoka et al (1994). They found that CO coordination to b-NOS is biphasic in the absence of substrate.…”

Section: Discussionmentioning

confidence: 72%

Interactions between Substrate Analogues and Heme Ligands in Nitric Oxide Synthase

1997

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“…On this basis, we ascribe both the 418 and 456 nm forms to low-spin compounds. The 418 nm absorbance was formerly shown to be present in a small fraction of nNOS(BH 4 +) (McMillan & Masters, 1993;Pufahl & Marletta, 1993;Matsuoka et al, 1994). By comparison with cytochrome P450, it has been ascribed (Matsuoka et al, 1994) to low-spin hexacoordinate heme, with a weakly bound ligand, most likely H 2 O, at the sixth coordination site (Poulos et al, 1986).…”

Section: High-and Low-spin Heme Structures In Bh 4 -Deficient Nnosmentioning

confidence: 99%

“…The transition of the small fraction of hexacoordinate lowspin heme, present in native nNOS, upon addition of Arg to the high-spin pentacoordinate form is well-documented (McMillan & Masters, 1993, 1995Matsuoka et al, 1994;Gerber & Ortiz de Montillano, 1995;Roman et al, 1995) and has its counterpart in the low-to high-spin transition observed after the addition of substrate to cytochrome P450 (Schenkman et al, 1967;Dawson & Sono, 1987). However, whereas NOS is already largely high-spin in the absence of Arg, low-spin heme predominates in most cytochrome P450s.…”

Section: Low-to High-spin Conversionmentioning

confidence: 99%

Tetrahydrobiopterin-Free Neuronal Nitric Oxide Synthase: Evidence for Two Identical Highly Anticooperative Pteridine Binding Sites

et al. 1996

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The properties of neuronal nitric oxide synthase containing one tetrahydrobiopterin (BH4) per dimer [nNOS(BH4+)] were compared to those of the BH4-free enzyme [nNOS(BH4-)]. The stimulation by BH4 of the formation of L-citrulline at the expense of H2O2 production unambiguously demonstrated that BH4 is essential in coupling reductive oxygen activation to Arg oxidation. The clear difference between the Stokes radii of nNOS(BH4-) and nNOS(BH4+) indicates that the introduction of one BH4 per dimer significantly changes the enzyme structure. Whereas the heme in nNOS(BH4+) was primarily high-spin, nNOS(BH4-) contained mainly low-spin heme. This was slowly converted into the high-spin form with Arg and/or BH4, with a rate that was independent of the concentration of either compound. Dithiothreitol inhibited the Arg/BH4-induced spin conversion by stabilizing low-spin heme. Formation of high-spin heme, with rates varying from 0.04 to 0.4 min-1, always correlated to an equally fast increase in activity. Radioligand binding studies showed the rapid association (within 20 s) of BH4 to nNOS(BH4-), but not to nNOS(BH4+), after preincubation with Arg. Complete and monophasic dissociation of radioligand occurred in the presence of excess unlabeled BH4, demonstrating the exchangeability of high-affinity bound BH4. Studies of the association of NG-nitro-L-arginine (L-NNA) to nNOS(BH4+) revealed that excess BH4 increased the amount of bound L-NNA 2-fold. Most of the binding data are explained by a model in which nNOS dimers accommodate two identical BH4- and Arg/L-NNA-binding sites, with cooperativity between Arg- and BH4-binding and anticooperativity between the BH4-binding sites.

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“…Quite recently, nNOS was produced in yeast (Black & Ortiz de Montellano, 1995) and Escherichia coli (Counts Gerber & Ortiz de Montellano, 1995;Roman et al, 1995) with excellent expression levels, as high as 25 mg of NOS per liter of culture. The problem of the heterologous expression of mNOS appears more difficult as this enzyme presents an added problem in that, as a result of calmodulin being tightly bound, the flow of electrons from NADPH to the heme is less well regulated (Cho et al, 1992;Abu-Soud et al, 1994;Matsuoka et al, 1994). This means that during the expression of mNOS, O 2 •-, ‚NO, and other products resulting from cascades involving these two species can be produced, all of which are potentially lethal to the cell.…”

mentioning

confidence: 99%

Expression in Yeast and Purification of Functional Macrophage Nitric Oxide Synthase. Evidence for Cysteine-194 As Iron Proximal Ligand

et al. 1996

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Mouse macrophage NO-synthase (mNOS) was expressed in a unique yeast-based system by using a three-step procedure which allows yeast growth and NOS expression to be uncoupled. Despite cytotoxic effects related to mNOS expression, levels of catalytically active enzyme up to 0.5 mg of protein per 5 L of culture was obtained after purification. Its electrophoretic, spectroscopic [lambda max = 446 nm for its Fe(II)-CO complex], and catalytic properties were similar to those previously reported for mNOS purified from macrophages. Recombinant mNOS catalyzed the NADPH-dependent oxidation of L-arginine to citrulline (Km = 7 +/- 3 microM) as well as the reduction of cytochrome C by NADPH [Km = 34 +/- 8 microM and Vm = 25 +/- 5 mumol min-1 (mg of protein-1)]. Two mutants of mNOS in which Cys 194 was replaced with either serine or histidine were constructed and expressed in the same yeast strain at a level higher than that of the wild type protein, as they appear less toxic for the host. Both mutants exhibited electrophoretic properties and activities toward cytochrome C reduction identical to those of wild type NOS. However, they were unable to catalyze the oxidation of L-arginine to citrulline and did not appear to bind heme (no appearance of peaks around 400 and 446 nm for the resting enzyme and its CO complex, respectively, in visible spectroscopy). These data provide the first experimental evidence in favor of previous suggestions that Cys 194 was the proximal iron ligand of mouse mNOS.

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L-arginine and calmodulin regulation of the heme iron reactivity in neuronal nitric oxide synthase.

Cited by 117 publications

References 34 publications

Interactions between Substrate Analogues and Heme Ligands in Nitric Oxide Synthase

Interactions between Substrate Analogues and Heme Ligands in Nitric Oxide Synthase

Tetrahydrobiopterin-Free Neuronal Nitric Oxide Synthase: Evidence for Two Identical Highly Anticooperative Pteridine Binding Sites

Expression in Yeast and Purification of Functional Macrophage Nitric Oxide Synthase. Evidence for Cysteine-194 As Iron Proximal Ligand

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