Restricting the conformational freedom of the neuronal nitric-oxide synthase flavoprotein domain reveals impact on electron transfer and catalysis

Dai, Yue; Haque, Mohammad Mahfuzul; Stuehr, Dennis J.

doi:10.1074/jbc.m117.777219

Cited by 7 publications

(5 citation statements)

References 44 publications

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“…As depicted in Scheme 1, FAD A 3 FMN A could occur in either open or closed conformation because studies have shown that the FMN domains are flexible, and this flexibility plays a vital role in electron transfer between the FMN, FAD, and heme. For example, restricting the freedom of the motion of the FMN domain greatly impairs the electron transfer of FMN as demonstrated elegantly in rat POR and the reductase domain of nNOS (27,28). As we have shown by 2D EM, the architecture of the BMR is a compact globular protein where the FMN and FAD domains are in proximity.…”

Section: Architecture Of Dimeric Cyp102a1mentioning

confidence: 87%

The full-length cytochrome P450 enzyme CYP102A1 dimerizes at its reductase domains and has flexible heme domains for efficient catalysis

Zhang¹,

Yokom

Shen³

et al. 2018

Journal of Biological Chemistry

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The cytochrome P450 enzyme CYP102A1 from is a highly efficient hydroxylase of fatty acids, and there is a significant interest in using CYP102A1 for biotechnological applications. Here, we used size-exclusion chromatography-multiangle light scattering (SEC-MALS) analysis and negative-stain EM to investigate the molecular architecture of CYP102A1. The SEC-MALS analysis yielded a homogeneous peak with an average molecular mass of 235 ± 5 kDa, consistent with homodimeric CYP102A1. The negative-stain EM of dimeric CYP102A1 revealed four distinct lobes, representing the two heme and two reductase domains. Two of the lobes were in close contact, whereas the other two were often observed apart and at the ends of a U-shaped configuration. The overall dimension of the dimer was ∼130 Å. To determine the identity of the lobes, we FLAG-tagged the N or C terminus of CYP102A1 to visualize additional densities in EM and found that anti-FLAG Fab could bind only the N-tagged P450. Single-particle analysis of this anti-Flag Fab-CYP102A1 complex revealed additional density in the N-terminally tagged heme domains, indicating that the heme domains appear flexible, whereas the reductase domains remain tightly associated. The effects of truncation on CYP102A1 dimerization, identification of cross-linked sites by peptide mapping, and molecular modeling results all were consistent with the dimerization of the reductase domain. We conclude that functional CYP102A1 is a compact globular protein dimerized at its reductase domains, with its heme domains exhibiting multiple conformations that likely contribute to the highly efficient catalysis of CYP102A1.

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Section: Architecture Of Dimeric Cyp102a1mentioning

confidence: 87%

The full-length cytochrome P450 enzyme CYP102A1 dimerizes at its reductase domains and has flexible heme domains for efficient catalysis

Zhang¹,

Yokom

Shen³

et al. 2018

Journal of Biological Chemistry

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“…We know that the chaperone Hsp90 participates in sGC maturation by bind-ing directly to apo-sGC␤. Its binding prevents apo-sGC␤ from forming a heme-free sGC␣/␤ heterodimer and also enables heme insertion into apo-sGC␤ in an ATP-dependent process (15,16). However, how heme is delivered to apo-sGC␤ remains unclear.…”

mentioning

confidence: 99%

GAPDH delivers heme to soluble guanylyl cyclase

Dai

Sweeny

Schlanger

et al. 2020

Journal of Biological Chemistry

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Soluble guanylyl cyclase (sGC) is a key component of NO–cGMP signaling in mammals. Although heme must bind in the sGC β1 subunit (sGCβ) for sGC to function, how heme is delivered to sGCβ remains unknown. Given that GAPDH displays properties of a heme chaperone for inducible NO synthase, here we investigated whether heme delivery to apo-sGCβ involves GAPDH. We utilized an sGCβ reporter construct, tetra-Cys sGCβ, whose heme insertion can be followed by fluorescence quenching in live cells, assessed how lowering cell GAPDH expression impacts heme delivery, and examined whether expressing WT GAPDH or a GAPDH variant defective in heme binding recovers heme delivery. We also studied interaction between GAPDH and sGCβ in cells and their complex formation and potential heme transfer using purified proteins. We found that heme delivery to apo-sGCβ correlates with cellular GAPDH expression levels and depends on the ability of GAPDH to bind intracellular heme, that apo-sGCβ associates with GAPDH in cells and dissociates when heme binds sGCβ, and that the purified GAPDH–heme complex binds to apo-sGCβ and transfers its heme to sGCβ. On the basis of these results, we propose a model where GAPDH obtains mitochondrial heme and then forms a complex with apo-sGCβ to accomplish heme delivery to sGCβ. Our findings illuminate a critical step in sGC maturation and uncover an additional mechanism that regulates its activity in health and disease.

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“…For instance, congenital dysplasia of the ENS leads to congenital megacolon (Hirschsprung disease) and primary achalasia, whereas neurodegenerative ENS alterations can lead to the disorders such as Alzheimer, Huntington, and Parkinson diseases[ 16 , 17 ]. The secondary alterations in the ENS also result in inflammatory infiltrates or immune system pathologies such as irritable bowel syndrome[ 18 ], idiopathic enteric gangliosidosis[ 19 ], paraneoplastic syndrome[ 20 ], slow transit constipation[ 21 ], severe acute pancreatitis[ 22 ], diabetes mellitus[ 23 , 24 ], and UC[ 25 ].…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, it could be concluded that as a messenger of information transmission between the NOS-positive neurons and GI SMCs, increased NO can reduce the Ca 2+ influx and directly promotes smooth muscle relaxation. In addition, studies have confirmed that NO can inhibit muscle contraction by inhibiting the release of excitatory transmitters[ 23 , 36 ]. Therefore, NO-mediated reduced contractility of the intestinal smooth muscle might be one of the important mechanisms contributing to colonic dysmotility[ 9 , 37 ].…”

Section: Discussionmentioning

confidence: 99%

Involvement of nitrergic neurons in colonic motility in a rat model of ulcerative colitis

Li¹,

Li²,

Jin³

et al. 2022

WJG

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BACKGROUND The mechanisms underlying gastrointestinal (GI) dysmotility with ulcerative colitis (UC) have not been fully elucidated. The enteric nervous system (ENS) plays an essential role in the GI motility. As a vital neurotransmitter in the ENS, the gas neurotransmitter nitric oxide (NO) may impact the colonic motility. In this study, dextran sulfate sodium (DSS)-induced UC rat model was used for investigating the effects of NO by examining the effects of rate-limiting enzyme nitric oxide synthase (NOS) changes on the colonic motility as well as the role of the ENS in the colonic motility during UC. AIM To reveal the relationship between the effects of NOS expression changes in NOS-containing nitrergic neurons and the colonic motility in a rat UC model. METHODS Male rats ( n = 8/each group) were randomly divided into a control (CG), a UC group (EG1), a UC + thrombin derived polypeptide 508 trifluoroacetic acid (TP508TFA; an NOS agonist) group (EG2), and a UC + NG-monomethyl-L-arginine monoacetate (L-NMMA; an NOS inhibitor) group (EG3). UC was induced by administering 5.5% DSS in drinking water without any other treatment (EG1), while the EG2 and EG3 were gavaged with TP508 TFA and L-NMMA, respectively. The disease activity index (DAI) and histological assessment were recorded for each group, whereas the changes in the proportion of colonic nitrergic neurons were counted using immunofluorescence histochemical staining, Western blot, and enzyme linked immunosorbent assay, respectively. In addition, the contractile tension changes in the circular and longitudinal muscles of the rat colon were investigated in vitro using an organ bath system. RESULTS The proportion of NOS-positive neurons within the colonic myenteric plexus (MP), the relative expression of NOS, and the NOS concentration in serum and colonic tissues were significantly elevated in EG1, EG2, and EG3 compared with CG rats. In UC rats, stimulation with agonists and inhibitors led to variable degrees of increase or decrease for each indicator in the EG2 and EG3. When the rats in EGs developed UC, the mean contraction tension of the colonic smooth muscle detected in vitro was higher in the EG1, EG2, and EG3 than in the CG group. Compared with the EG1, the contraction amplitude and mean contraction tension of the circular and longitudinal muscles of the colon in the EG2 and EG3 were enhanced and attenuated, respectively. Thus, during UC, regulation of the expression of NOS within the MP improved the intestinal motility, thereby favoring the recovery of intestinal functions. CONCLUSION In UC rats, an increased number of nitrergic neurons in the colonic MP leads to the attenuation of colonic motor function. To intervene NOS activity might modulate the function of nitrergic neurons in the colonic MP and prevent colonic motor dysfunction. These resu...

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Restricting the conformational freedom of the neuronal nitric-oxide synthase flavoprotein domain reveals impact on electron transfer and catalysis

Cited by 7 publications

References 44 publications

The full-length cytochrome P450 enzyme CYP102A1 dimerizes at its reductase domains and has flexible heme domains for efficient catalysis

The full-length cytochrome P450 enzyme CYP102A1 dimerizes at its reductase domains and has flexible heme domains for efficient catalysis

GAPDH delivers heme to soluble guanylyl cyclase

Involvement of nitrergic neurons in colonic motility in a rat model of ulcerative colitis

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