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

Zheng, Huayu; Li, Jinghui; Feng, Changjian

doi:10.1002/1873-3468.13870

Cited by 6 publications

(2 citation statements)

References 41 publications

(67 reference statements)

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“…We observed several cytoplasmic and mitochondrial heat shock proteins in our analysis ( Figure 6; Table S6 ). In addition to their well-established roles in protein folding, quality control, and the stress response 79 , HSPs are also involved in iron/sulfur cluster biogenesis, important for heme biosynthesis 80 ; and in facilitating catalytic activities including intra-protein electron transfer to heme cofactors 81 . Of note, HSPs have also been directly implicated in heme insertion into hemoproteins.…”

Section: Discussionmentioning

confidence: 99%

Depletion Assisted Hemin Affinity (DAsHA) Proteomics Reveals an Expanded Landscape of Heme Binding Proteins

Kim

Moore

Mestre-Fos

et al. 2022

Preprint

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Heme b (iron protoporphyrin IX) plays important roles in biology as a metallocofactor and signaling molecule. However, the targets of heme signaling and the network of proteins that mediate the exchange of heme from sites of synthesis or uptake to heme dependent or regulated proteins are poorly understood. Herein, we describe a quantitative mass spectrometry-based chemoproteomics strategy to identify exchange labile hemoproteins in human embryonic kidney HEK293 cells that may be relevant to heme signaling and trafficking. The strategy involves depleting endogenous heme with the heme biosynthetic inhibitor succinylacetone (SA), leaving putative heme binding proteins in their apo-state, followed by the capture of those proteins using hemin-agarose resin and finally elution and identification by mass spectrometry. By identifying only those proteins that interact with high specificity to hemin-agarose relative to control beaded agarose in a SA-dependent manner, we have expanded the number of proteins and ontologies that may be involved in binding and buffering labile heme or are targets of heme signaling. Notably, these include proteins involved in chromatin remodeling, DNA damage response, RNA splicing, cytoskeletal organization and vesicular trafficking, many of which have been associated with heme through complimentary studies published recently. Taken together, these results provide support for the emerging role for heme in an expanded set of cellular processes from genome integrity to protein trafficking and beyond.

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Section: Discussionmentioning

confidence: 99%

Depletion Assisted Hemin Affinity (DAsHA) Proteomics Reveals an Expanded Landscape of Heme Binding Proteins

Kim

Moore

Mestre-Fos

et al. 2022

Preprint

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“…Furthermore, active NOS enzymes represent a conformational ensemble of dynamic interdomain interactions. The NOS enzyme is also regulated by extrinsic factors including proteins such as CaM and heat shock protein 90, − as well as posttranslational modifications such as phosphorylation. , Moreover, the intrinsic regulatory elements are inherently disordered. For example, in the crystal structure of a CaM-free nNOS reductase, only a short helical portion was observed for the AR, and half of the CT remained unresolved.…”

Section: Introductionmentioning

confidence: 99%

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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Nitric Oxide in Human Physiology

Mohammed

Zuckerbraun

2022

Carbon Monoxide in Drug Discovery

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

Cited by 6 publications

References 41 publications

Depletion Assisted Hemin Affinity (DAsHA) Proteomics Reveals an Expanded Landscape of Heme Binding Proteins

Depletion Assisted Hemin Affinity (DAsHA) Proteomics Reveals an Expanded Landscape of Heme Binding Proteins

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

Nitric Oxide in Human Physiology

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