title: Heme-dependent activation of neuronal nitric oxide synthase by cytosol is due to an Hsp70-dependent, thioredoxin-mediated thiol-disulfide interchange in the heme/substrate binding cleft.
abstract: We have reported that heme-dependent activation of apo-neuronal nitric oxide synthase (apo-nNOS) to the active holo-enzyme dimer is dependent upon factors present in reticulocyte lysate and other cytosols. Here, we find that both Hsp70 and thioredoxin are components of the activation system. The apo-nNOS activating activity of reticulocyte lysate is retained in a pool of fractions containing Hsp70 that elute from DE52 prior to Hsp90. All of the activating activity and 20-30% of the Hsp70 elute in the flow-through fraction upon subsequent ATP-agarose chromatography. Apo-nNOS activation by this flow-through fraction is inhibited by pifithrin-μ, a small molecule inhibitor of Hsp70, suggesting that a non-ATP-binding form of Hsp70 is involved in heme-dependent apo-nNOS activation. Previous work has shown that apo-nNOS can be activated by thiol-disulfide exchange, and we show substantial activation with a small molecule dithiol modeled on the active motifs of thioredoxin and protein disulfide isomerase. Further fractionation of the ATP-agarose flow-through on Sephacryl S-300 separates free thioredoxin from apo-nNOS activating activity, Hsp70, and a small amount of thioredoxin, all of which are eluted throughout the macromolecular peak. Incubation of apo-nNOS with the macromolecular fraction in combination either with the thioredoxin-containing fraction or with purified recombinant human thioredoxin restores full heme-dependent activating activity. This supports a model in which Hsp70 binding to apo-nNOS stabilizes an open state of the heme/substrate binding cleft to facilitate thioredoxin access to the active site cysteine that coordinates with heme iron, permitting heme binding and dimerization to the active enzyme.
authors.affiliation: Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Morishima
authors.forename: Miranda
authors.initials: M
authors.lastname: Lau
authors.forename: Hwei-Ming
authors.initials: HM
authors.lastname: Peng
authors.forename: Yoshinari
authors.initials: Y
authors.lastname: Miyata
authors.forename: Jason E
authors.initials: JE
authors.lastname: Gestwicki
authors.forename: William B
authors.initials: WB
authors.lastname: Pratt
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Osawa
mesh_terms: D000818:Animals; D001051:Apoenzymes; D001779:Blood Coagulation Factors / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism*; D004789:Enzyme Activation; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D011485:Protein Binding; D016601:RNA-Binding Proteins; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes; D012269:Ribosomal Proteins; D012685:Sepharose / Q000031:analogs & derivatives; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism*
chemical_list: C030548:ATP-sepharose; D001051:Apoenzymes; D001779:Blood Coagulation Factors; D004220:Disulfides; D018840:HSP70 Heat-Shock Proteins; D016601:RNA-Binding Proteins; C104946:RPL29 protein, human; D012269:Ribosomal Proteins; D013438:Sulfhydryl Compounds; D006418:Heme; D013879:Thioredoxins; D012685:Sepharose; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat