Catalytic Intermediates of Inducible Nitric-oxide Synthase Stabilized by the W188H Mutation*

Sabat, Joseph; Egawa, Tsuyoshi; Lu, Chong‐Dao; Stuehr, Dennis J.; Gerfen, Gary J.; Rousseau, Denis L.; Yeh, Syun Ru

doi:10.1074/jbc.m112.403238

Cited by 14 publications

(21 citation statements)

References 56 publications

(36 reference statements)

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“…The lower mesohaem content observed in W188Hoxy seems to be an effect of the Trp to His mutation per se. This could be related to the recently noted misalignment between the imidazole ring of His 188 and the porphyrin ring as opposed to the native arrangement of the indole group of Trp 188 [11]. Conceivably, this structural restriction may affect cellular haem insertion during protein maturation.…”

Section: Resultsmentioning

confidence: 99%

“…Of particular interest was to examine the formation (or absence) of the inert enzyme species P whose exact nature remains elusive [3, 11]. L-Arg hydroxylation reactions of mesohaem-containing iNOSoxy could be best fit to a two-exponential model A →B →C, with Fe(II), Fe(II)-O 2 and Fe(III) as the only detectable species (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…5). We investigated whether reversal of the midpoint potential in W188Hoxy-mesohaem impacts the formation and stability of species P, which forms downstream of the Fe-O 2 species [3, 11]. L-Arg hydroxylation reactions of mesohaem-containing W188Hoxy were fit to a three-exponential model A →B →C→D, with Fe(II), Fe(II)-O 2 , a presumptive species P with absorption maximum at 411 nm and Fe(III) as the only detectable species (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We present a comprehensive examination of the effect of porphyrin replacement on the following features: Structure of wild type iNOSoxy-mesohaem, haem midpoint potential, enzyme oligomeric state, substrate binding, FeO 2 stability, kinetics of haem transitions during catalysis, extent of product formation, reactivity of the Fe-NO complex and NO synthesis. This is the first study to: i) Provide a crystal structure of inducible NOS substituted with mesohaem; ii) Establish the differential influence of structural and electronic components on the kinetic behaviour of iNOS and iii) Demonstrate that the formation of inert enzyme species P formed by iNOS mutant W188H [3, 11] is heavily influenced by an increased midpoint potential of the haem centre.…”

Section: Introductionmentioning

confidence: 99%

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Dissecting structural and electronic effects in inducible nitric oxide synthase

Hannibal

Page

Haque

et al. 2015

Biochemical Journal

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Nitric oxide synthases (NOS) are haem-thiolate enzymes that catalyse the conversion of L-Arginine (LArg) into NO and citrulline. Inducible NOS (iNOS) is responsible for delivery of NO in response to stressors during inflammation. The catalytic performance of iNOS is proposed to rely mainly on the haem midpoint potential and the ability of the substrate L-Arg to provide an H-bond for oxygen activation (O-O scission). We present a comparative study of native iNOS versus iNOS-mesohaem, and investigate the formation of a low-spin ferric haem-aquo or -hydroxo species (P) in iNOS mutant W188H substituted with mesohaem. iNOS-mesohaem and W188H-mesohaem were stable and dimeric, and presented substrate-binding affinities comparable to their native counterparts. Single turnover reactions catalysed by iNOSoxy with LArg (first reaction step) or N-hydroxyarginine (second reaction step) showed that mesohaem substitution triggered faster rates of FeIIO2 conversion and altered other key kinetic parameters. We elucidated the first crystal structure of a NOS substituted with mesohaem and found essentially identical features compared to the structure of iNOS carrying native haem. This facilitated the dissection of structural and electronic effects. Mesohaem substitution substantially reduced the build-up of species P in W188H iNOS during catalysis, thus increasing its proficiency toward NO synthesis. The marked structural similarities of iNOSoxy containing native haem or mesohaem indicate that the kinetic behaviour observed in mesohaem-substituted iNOS is most heavily influenced by electronic effects rather than structural alterations.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Dissecting structural and electronic effects in inducible nitric oxide synthase

Hannibal

Page

Haque

et al. 2015

Biochemical Journal

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“…This transfer is facilitated by calmodulin, which controls the intersubunit binding of FMN to heme. Heme is a switchpoint during the synthesis of NO • from arginine/N-hydroxy-l-arginine under consumption of oxygen and NADPH and the release of l-citrulline (see Sabat et al 2013). Since NO, as a diffusible molecule, can act as a retrograde diffusible messenger in the brain and can promote long-term potentiation through glutamatergic transmission, it appears to be promising to study the interrelationship between NO • production and channel gating, resulting in depolarization via Nav channels followed by activation of cell Cav channels as described (Figueroa et al 2007) (Fig.…”

Section: Discussionmentioning

confidence: 99%

Cryptochrome in Sponges

Müller

Schröder

Markl

et al. 2013

J Histochem Cytochem.

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SummarySponges (phylum: Porifera) react to external light or mechanical signals with contractile or metabolic reactions and are devoid of any nervous or muscular system. Furthermore, elements of a photoreception/phototransduction system exist in those animals. Recently, a cryptochrome-based photoreceptor system has been discovered in the demosponge. The assumption that in sponges the siliceous skeleton acts as a substitution for the lack of a nervous system and allows light signals to be transmitted through its glass fiber network is supported by the findings that the first spicules are efficient light waveguides and the second sponges have the enzymatic machinery for the generation of light. Now, we have identified/ cloned in Suberites domuncula two additional potential molecules of the sponge cryptochrome photoreception system, the guanine nucleotide-binding protein β subunit, related to β-transducin, and the nitric oxide synthase (NOS)-interacting protein. Cryptochrome and NOSIP are light-inducible genes. The studies show that the NOS inhibitor L-NMMA impairs both morphogenesis and motility of the cells. Finally, we report that the function of primmorphs to produce reactive nitrogen species can be abolished by a NOS inhibitor. We propose that the sponge cryptochrome-based photoreception system, through which photon signals are converted into radicals, is coupled to the NOS apparatus. ( J Histochem Cytochem 61:814-832, 2013)

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Oxygen activation in NO synthases: evidence for a direct role of the substrate

et al. 2016

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Nitric oxide ( NO ) and the other reactive nitrogen species ( RNOS ) play crucial patho‐physiological roles at the interface of oxidative stress and signalling processes. In mammals, the NO synthases ( NOS s) are the source of these reactive nitrogen species, and so to understand the precise biological role of RNOS and NO requires elucidation of the molecular functioning of NOS . Oxygen activation, which is at the core of NOS catalysis, involves a sophisticated sequence of electron and proton transfers. While electron transfer in NOS has received much attention, the proton transfer processes has been scarcely investigated. Here, we report an original approach that combines fast‐kinetic techniques coupled to resonance Raman spectroscopy with the use of synthetic analogues of NOS substrate. We characterise Fe II ‐O 2 reaction intermediates in the presence of L‐arginine (Arg), alkyl‐ and aryl‐guanidines. The presence of new reaction intermediates, such as ferric haem‐peroxide, that was formerly postulated, was tracked by analysing the oxygen activation reaction at different times and with different excitation wavelengths. Our results suggest that Arg is not a proton donor, but indirectly intervenes in oxygen activation mechanism by modulating the distal H‐bond network and, in particular, by tuning the position and the role of the distal water molecule. This report supports a catalytic model with two proton transfers in step 1 (Arg hydroxylation) but only one proton transfer in step 2 (N ω ‐hydroxy‐L‐arginine oxidation).

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Catalytic Intermediates of Inducible Nitric-oxide Synthase Stabilized by the W188H Mutation*

Cited by 14 publications

References 56 publications

Dissecting structural and electronic effects in inducible nitric oxide synthase

Dissecting structural and electronic effects in inducible nitric oxide synthase

Cryptochrome in Sponges

Oxygen activation in NO synthases: evidence for a direct role of the substrate

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