Characterization of bovine endothelial nitric oxide synthase as a homodimer with down-regulated uncoupled NADPH oxidase activity: tetrahydrobiopterin binding kinetics and role of haem in dimerization

List, Barbara M.; Klösch, Burkhard; Völker, Christof; Gorren, Antonius C.F.; Sessa, William C.; Werner, Ernst R.; Kukovetz, W. R.; Schmidt, Kurt; Mayer, Bernd

doi:10.1042/bj3230159

Cited by 142 publications

(120 citation statements)

References 59 publications

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“…As shown in the upper panel of Fig. 1, an anaerobic sample of ferric eNOS containing H4B displayed a broad Soret absorbance peak centered near 400 nm with flavin absorbance peaks ranging from 445 to 550 nm, consistent with earlier reports (8,9,23). Adding NADPH in the absence of bound CaM caused flavin reduction as judged by the disappearance of the flavin visible absorption bands.…”

Section: Extent and Kinetics Of Flavin And Hemesupporting

confidence: 75%

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Electron Transfer, Oxygen Binding, and Nitric Oxide Feedback Inhibition in Endothelial Nitric-oxide Synthase

Abu-Soûd

Ichimori

Presta

et al. 2000

Journal of Biological Chemistry

116

118

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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...

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Section: Extent and Kinetics Of Flavin And Hemesupporting

confidence: 75%

“…A comparison of published steady-state rates shows that eNOS is about four to eight times slower than either nNOS or iNOS (7)(8)(9)(10)(11)(12)(13)(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.…”

mentioning

confidence: 99%

Electron Transfer, Oxygen Binding, and Nitric Oxide Feedback Inhibition in Endothelial Nitric-oxide Synthase

Abu-Soûd

Ichimori

Presta

et al. 2000

Journal of Biological Chemistry

116

118

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“…Although the dissociation rate constants for BH 4 release from NOS at neutral pH are slow, reported as 0.01-0.3 min Ϫ1 (41)(42)(43), BH 4 release would likely be greatly accelerated under the acidotic conditions of the ischemic heart. Furthermore, oxidation of BH 4 can occur to both free and NOS-bound biopterin with rapid loss of BH 4 and XPH 2 formation (J.L.Z., R.B., W. Chen, and A.J.C., unpublished results).…”

Section: Discussionmentioning

confidence: 99%

Myocardial ischemia results in tetrahydrobiopterin (BH ₄ ) oxidation with impaired endothelial function ameliorated by BH ₄

Dumitrescu¹,

Biondi

Xia³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

183

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Coronary vasodilation is impaired in the postischemic heart with a loss of endothelial nitric oxide synthase (eNOS) activity, but the mechanisms underlying ischemia-induced eNOS dysfunction are not understood. For nitric oxide (NO) synthesis, eNOS requires the redox-sensitive cofactor tetrahydrobiopterin (BH 4); however, the role of BH 4 in ischemia-induced endothelial dysfunction remains unknown. Therefore, isolated rat hearts were subjected to varying durations of ischemia, and the alterations in NOS-dependent vasodilation were measured and correlated with assays of eNOS activity and cardiac BH 4 concentrations. Ischemia timedependently decreased cardiac BH 4 content with 85, 95, or 97% irreversible degradation after 30, 45, or 60 min of ischemia, respectively. Paralleling the decreases in BH 4, reductions of eNOS activity were seen of 58, 86, or 92%, and NOS-derived superoxide production was greatly increased. Addition of 10 M BH4 enhanced eNOS activity in nonischemic hearts and partially restored activity after ischemia. It also suppressed NOS-derived superoxide production. Impaired coronary flow during postischemic reperfusion was improved by BH 4 infusion. Thus, BH4 depletion contributes to postischemic eNOS dysfunction, and BH 4 treatment is effective in partial restoration of endothelium-dependent coronary flow. Supplementation of BH 4 may therefore be an important therapeutic approach to reverse endothelial dysfunction in postischemic tissues.ischemia reperfusion injury ͉ nitric oxide ͉ nitric oxide synthase uncoupling ͉ superoxide ͉ vascular function N itric oxide synthase (NOS) converts L-arginine and O 2 to nitric oxide (NO) and L-citrulline. This enzymatic process consumes NADPH and requires Ca 2ϩ /calmodulin, flavin adenine dinucleotide, flavin mononucleotide, and tetrahydrobiopterin (BH 4 ) as NOS cofactors. Endothelial NO synthase (eNOS) contributes to the regulation of vasomotor tone and blood pressure by producing NO that activates soluble guanylate cyclase in vascular smooth muscle, resulting in vasorelaxation (1-3).Endothelial dysfunction is a prognostic marker of cardiovascular disease (4). It has been suggested that limited availability of BH 4 contributes to eNOS dysfunction in hypercholesterolemia, diabetes, atherosclerosis, hypertension, and heart failure (5-9). It was also observed previously that eNOS function is impaired in ischemic hearts (10). In vivo coronary artery occlusion triggers endothelial dysfunction and decreased eNOSdependent vasoreactivity, although reactivity is preserved to exogenous NO (11,12). Endothelial-dependent coronary vasoreactivity is impaired in hearts subjected to periods of global ischemia and reperfusion (10). Endothelium-dependent vasodilators induce a relatively high increase in coronary flow in control hearts or in those made ischemic for short times, but longer periods of ischemia result in an abrupt decline in vasodilatory response.In addition to impairing eNOS-mediated NO formation, BH 4 depletion may have additional detrimental effects in postischem...

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“…Recently, it has been shown that dimerization is required for catalytic activity in all 3 NOS isoforms (1)(2)(3)(4)(5). The process of dimerization appears to depend on the presence of the cofactors heme and tetrahydrobiopterin as well as the enzyme sub-strate, L-arginine.…”

Section: Introductionmentioning

confidence: 99%

Use of Chimeric Forms of Neuronal Nitric‐Oxide Synthase as Dominant Negative Mutants

Phung

Black

1999

IUBMB Life

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SummaryBecause the functional form of neuronal nitric-oxide synthase (nNOS) is a homodimer, we investigated whether we could disrupt dimer formation with inactive nNOS chimeras acting as dominant negative mutants. To test this hypothesis, we either expressed the heme and reductase regions of rat nNOS as single domains or produced fusion proteins between the rat nNOS heme domain and various other electron-shuttling proteins. A dominant negative potential of these constructs was demonstrated by their ability to reduce NOS activity when transfected into a cell line stably expressing rat nNOS. In the presence of these nNOS mutant proteins, cellular levels of inactive nNOS monomers were signi cantly increased, indicating that their mechanism of action is through the disruption of nNOS dimer formation. These dominant negative mutants should prove valuable in analyzing the role of nNOS in biological systems. IUBMB Life, 48: 333-338, 1999

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Characterization of bovine endothelial nitric oxide synthase as a homodimer with down-regulated uncoupled NADPH oxidase activity: tetrahydrobiopterin binding kinetics and role of haem in dimerization

Cited by 142 publications

References 59 publications

Electron Transfer, Oxygen Binding, and Nitric Oxide Feedback Inhibition in Endothelial Nitric-oxide Synthase

Electron Transfer, Oxygen Binding, and Nitric Oxide Feedback Inhibition in Endothelial Nitric-oxide Synthase

Myocardial ischemia results in tetrahydrobiopterin (BH ₄ ) oxidation with impaired endothelial function ameliorated by BH ₄

Use of Chimeric Forms of Neuronal Nitric‐Oxide Synthase as Dominant Negative Mutants

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Characterization of bovine endothelial nitric oxide synthase as a homodimer with down-regulated uncoupled NADPH oxidase activity: tetrahydrobiopterin binding kinetics and role of haem in dimerization

Cited by 142 publications

References 59 publications

Electron Transfer, Oxygen Binding, and Nitric Oxide Feedback Inhibition in Endothelial Nitric-oxide Synthase

Electron Transfer, Oxygen Binding, and Nitric Oxide Feedback Inhibition in Endothelial Nitric-oxide Synthase

Myocardial ischemia results in tetrahydrobiopterin (BH 4 ) oxidation with impaired endothelial function ameliorated by BH 4

Use of Chimeric Forms of Neuronal Nitric‐Oxide Synthase as Dominant Negative Mutants

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Myocardial ischemia results in tetrahydrobiopterin (BH ₄ ) oxidation with impaired endothelial function ameliorated by BH ₄