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

ABSTRACT: 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 Hsp90? was used to investigate whether (and if so, how) Hsp90 affects the FMN-heme interdomain electron transfer (IET) step in NOS. Hsp90? 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.

SUBMITTER: Zheng H

PROVIDER: S-EPMC7722166 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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

Zheng Huayu H Li Jinghui J Feng Changjian C

FEBS letters 20200704 17

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 abo ...[more]

PMID: 32573772

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Project description:Previous biochemical studies of nitric oxide synthase enzymes (NOSs) were conducted in diluted solutions. However, the intracellular milieu where the proteins perform their biological functions is crowded with macromolecules. The effect of crowding on the electron transfer kinetics of multidomain proteins is much less understood. Herein, we investigated the effect of macromolecular crowding on the FMN-heme intraprotein interdomain electron transfer (IET), an obligatory step in NOS catalysis. A noticeable increase in the IET rate in the bidomain oxygenase/FMN (oxyFMN) and the holoprotein of human inducible NOS (iNOS) was observed upon addition of Ficoll 70 in a nonsaturable manner. Additionally, the magnitude of IET enhancement for the holoenzyme is much higher than that that of the oxyFMN construct. The crowding effect is also evident at different ionic strengths. Importantly, the enhancing extent is similar for the iNOS oxyFMN protein with added Ficoll 70 and Dextran 70 that give the same solution viscosity, showing that specific interactions do not exist between the NOS protein and the crowder. Moreover, the population of the docked FMN-heme state is significantly increased upon addition of Ficoll 70 and the fluorescence lifetime values do not correspond to those in the absence of Ficoll 70. The steady-state cytochrome c reduction by the holoenzyme is noticeably enhanced by the crowder, while the ferricyanide reduction is unchanged. The NO production activity of the iNOS holoenzyme is stimulated by Ficoll 70. The effect of macromolecular crowding on the kinetics can be rationalized on the basis of the excluded volume effect, with an entropic origin. The intraprotein electron transfer kinetics, fluorescence lifetime, and steady-state enzymatic activity results indicate that macromolecular crowding modulates the NOS electron transfer through multiple pathways. Such a mechanism should be applicable to electron transfer in other multidomain redox proteins.

Dataset Information

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

Publications

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

Similar Datasets

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