Role of an isoform‐specific residue at the calmodulin–heme (<scp>NO</scp> synthase) interface in the <scp>FMN</scp> – heme electron transfer

Li, Jinghui; Zheng, Huayu; Wang, Wei; Miao, Yubin; Sheng, Yinghong; Feng, Changjian

doi:10.1002/1873-3468.13158

Cited by 7 publications

(4 citation statements)

References 36 publications

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“…Also of note is that the CaM-NOS interacting/docking sites identified from previous mutational studies , yield CaM-nNOS cross-links with >40 Å distance, e.g., 94–466, 94–469 (Table ). The wide range of cross-links, in particular at the docking interfaces, indicate a conformation sampling mechanism in which extensive motions at the protein interfaces give a range of dynamic, transient donor–acceptor complexes, only a subset of which are IET-competent .…”

Section: Resultsmentioning

confidence: 97%

Mapping the Intersubunit Interdomain FMN-Heme Interactions in Neuronal Nitric Oxide Synthase by Targeted Quantitative Cross-Linking Mass Spectrometry

Jiang,

Wan,

Zhang

et al. 2024

Biochemistry

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Nitric oxide synthase (NOS) in mammals is a family of multidomain proteins in which interdomain electron transfer (IET) is controlled by domain–domain interactions. Calmodulin (CaM) binds to the canonical CaM-binding site in the linker region between the FMN and heme domains of NOS and allows tethered FMN domain motions, enabling an intersubunit FMN-heme IET in the output state for NO production. Our previous cross-linking mass spectrometric (XL MS) results demonstrated site-specific protein dynamics in the CaM-responsive regions of rat neuronal NOS (nNOS) reductase construct, a monomeric protein [Jiang et al., Biochemistry, 2023, 62, 2232–2237]. In this work, we have extended our combined approach of XL MS structural mapping and AlphaFold structural prediction to examine the homodimeric nNOS oxygenase/FMN (oxyFMN) construct, an established model of the NOS output state. We employed parallel reaction monitoring (PRM) based quantitative XL MS (qXL MS) to assess the CaM-induced changes in interdomain dynamics and interactions. Intersubunit cross-links were identified by mapping the cross-links onto top AlphaFold structural models, which was complemented by comparing their relative abundances in the cross-linked dimeric and monomeric bands. Furthermore, contrasting the CaM-free and CaM-bound nNOS samples shows that CaM enables the formation of the intersubunit FMN-heme docking complex and that CaM binding induces extensive, allosteric conformational changes across the NOS regions. Moreover, the observed cross-links sites specifically respond to changes in ionic strength. This indicates that interdomain salt bridges are responsible for stabilizing and orienting the output state for efficient FMN-heme IET. Taken together, our targeted qXL MS results have revealed that CaM and ionic strength modulate specific dynamic changes in the CaM/FMN/heme complexes, particularly in the context of intersubunit interdomain FMN-heme interactions.

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Section: Resultsmentioning

confidence: 97%

Mapping the Intersubunit Interdomain FMN-Heme Interactions in Neuronal Nitric Oxide Synthase by Targeted Quantitative Cross-Linking Mass Spectrometry

Jiang,

Wan,

Zhang

et al. 2024

Biochemistry

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“…4). Such polarity reversal approach has been used in study of charge interactions in the interdomain interfaces in rat nNOS [40] and human iNOS [8]. The IET rate for the sample containing Hsp90 K585E and nNOS D552K mutants (32 ± 1 s −1 ) exhibits modest recovery toward the wt level (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…CaM binding to the linker between the reductase and oxygenase domains of NOS is required for release of the FMN domain from the NADPH/FAD domain, and the subsequent large‐scale shuttling motion of the FMN domain to transport the NADPH‐derived electron to the catalytic heme domain (where NO production takes place) [4,5]. Besides its binding to the canonical linker, CaM docks to the NOS heme domain [6,7], which is mediated by formation of salt bridges [8]. Facilitating the interdomain FMN/heme alignment by CaM is essential for the functions of all the three NOS isoforms [9].…”

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confidence: 99%

Heat shock protein 90 enhances the electron transfer between the FMN and heme cofactors in neuronal nitric oxide synthase

Zheng

Feng

2020

FEBS Letters

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Heat shock protein 90 (Hsp90) is a key regulator of nitric oxide synthase (NOS) in vivo. Despite its functional importance, little is known about the underlying molecular mechanism. Here, purified dimeric human Hsp90α was used to investigate whether (and if so, how) Hsp90 affects the FMN–heme interdomain electron transfer (IET) step in NOS. Hsp90α increases the IET rate for rat neuronal NOS (nNOS) in a dose‐saturable manner, and a single charge‐neutralization mutation at conserved Hsp90 K585 abolishes the effect. The kinetic results with added Ficoll 70, a crowder, further indicate that Hsp90 enhances the FMN–heme IET through specific association with nNOS. The Hsp90‐nNOS docking models provide hints on the putative role of Hsp90 in constraining the available conformational space for the FMN domain motions.

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“…It is of current interest to study the discrete FMN -heme IET process [35,36], which is catalytically essential in the delivery of electrons required for O 2 activation in the heme domain and the subsequent NO synthesis [2]. We have determined the FMN -heme IET kinetics in wt, pSer 1412 and S1412E rat nNOS proteins at 460 nm using laser flash photolysis ( Figure 5), and the observed IET rates are listed in Table 1.…”

Section: Spectral Characterizations Of the Pser1412 Rat Nnos Proteinmentioning

confidence: 99%

Generation and characterization of functional phosphoserine-incorporated neuronal nitric oxide synthase holoenzyme

Zheng

et al. 2018

J Biol Inorg Chem

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Phosphorylation is an important pathway for regulation of nitric oxide synthase (NOS) at the posttranslational level. However, the molecular underpinnings of NOS regulation by phosphorylations remain unclear to date, mainly because of the problems in making a good amount of active phospho-NOS proteins. Herein, we have established a system in which recombinant rat nNOS holoprotein can be produced with site-specific incorporation of phosphoserine (pSer) at residue 1412, using a specialized bacterial host strain for pSerincorporation. The pSer1412 nNOS protein demonstrates UV-vis, far UV CD and fluorescence spectral properties that are identical to those of nNOS overexpressed in other bacterial strains. The protein is also functional, possessing normal NO production and NADPH oxidation activities in the presence of abundant substrate L-Arg. Conversely, the rate of FMN-heme interdomain electron transfer (IET) in pSer1412 nNOS is considerably lower than that of wild type (wt) nNOS, while the phosphomimetic S1142E mutant possesses similar electron transfer kinetics to that of wt. The successful incorporation and high yield of pSer1412 into rat nNOS and the significant change in the IET kinetics upon the phosphorylation demonstrate a highly useful method for incorporating native phosphorylation sites as a substantial improvement to commonly used phosphomimetics.

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Role of an isoform‐specific residue at the calmodulin–heme (NO synthase) interface in the FMN – heme electron transfer

Cited by 7 publications

References 36 publications

Mapping the Intersubunit Interdomain FMN-Heme Interactions in Neuronal Nitric Oxide Synthase by Targeted Quantitative Cross-Linking Mass Spectrometry

Mapping the Intersubunit Interdomain FMN-Heme Interactions in Neuronal Nitric Oxide Synthase by Targeted Quantitative Cross-Linking Mass Spectrometry

Heat shock protein 90 enhances the electron transfer between the FMN and heme cofactors in neuronal nitric oxide synthase

Generation and characterization of functional phosphoserine-incorporated neuronal nitric oxide synthase holoenzyme

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