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ABSTRACT: Heme insertion is key during maturation of soluble guanylyl cyclase (sGC) because it enables sGC to recognize NO and transduce its multiple biological effects. Although sGC is often associated with the 90-kDa heat shock protein (hsp90) in cells, the implications are unclear. The present study reveals that hsp90 is required to drive heme insertion into sGC and complete its maturation. We used a mammalian cell culture approach and followed heme insertion into transiently and endogenously expressed heme-free sGC. We used pharmacological hsp90 inhibitors, an ATP-ase inactive hsp90 mutant, and heme-dependent or heme-independent sGC activators as tools to decipher the role of hsp90. Our findings suggest that hsp90 complexes with apo-sGC, drives heme insertion through its inherent ATPase activity, and then dissociates from the mature, heme-replete sGC. Together, this improves our understanding of sGC maturation and reveals a unique means to control sGC activity in cells, and it has important implications for hsp90 inhibitor-based cancer therapy.
Proceedings of the National Academy of Sciences 07/2012; 109(32):12998-3003. · 9.68 Impact Factor
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ABSTRACT: In nitric-oxide synthases (NOSs), two flexible hinges connect the FMN domain to the rest of the enzyme and may guide its interactions with partner domains for electron transfer and catalysis. We investigated the role of the FMN-FAD/NADPH hinge in rat neuronal NOS (nNOS) by constructing mutants that either shortened or lengthened this hinge by 2, 4, and 6 residues. Shortening the hinge progressively inhibited electron flux through the calmodulin (CaM)-free and CaM-bound nNOS to cytochrome c, whereas hinge lengthening relieved repression of electron flux in CaM-free nNOS and had no impact or slowed electron flux through CaM-bound nNOS to cytochrome c. How hinge length influenced heme reduction depended on whether enzyme flavins were pre-reduced with NADPH prior to triggering heme reduction. Without pre-reduction, changing the hinge length was deleterious; with pre-reduction, the hinge shortening was deleterious, and hinge lengthening increased heme reduction rates beyond wild type. Flavin fluorescence and stopped-flow kinetic studies on CaM-bound enzymes suggested hinge lengthening slowed the domain-domain interaction needed for FMN reduction. All hinge length changes lowered NO synthesis activity and increased uncoupled NADPH consumption. We conclude that several aspects of catalysis are sensitive to FMN-FAD/NADPH hinge length and that the native hinge allows a best compromise among the FMN domain interactions and associated electron transfer events to maximize NO synthesis and minimize uncoupled NADPH consumption.
Journal of Biological Chemistry 06/2012; 287(36):30105-16. · 4.77 Impact Factor
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ABSTRACT: Maturation of NOS enzymes requires that they incorporate heme to become active, but how this cellular process occurs is unclear. We investigated a role for chaperone heat shock protein 90 (hsp90) in enabling heme insertion into the cytokine-inducible mouse NOS. We used macrophage cell line RAW 264.7 and human embryonic kidney HEK293T cells and studied insertion of native heme during iNOS expression and insertion of exogenous heme into preformed apo-iNOS. Pulldown experiments showed that the hsp90-iNOS complex was present in cells, but the extent of their association was inversely related to iNOS heme content. Hsp90 was primarily associated with apo-iNOS monomer and was associated 11-fold less with heme-containing iNOS monomer or dimer in cells. Kinetic studies showed that hsp90 dissociation occurred coincident with cellular heme insertion into apo-iNOS (0.8 h(-1)). The hsp90 inhibitor radicicol or coexpression of an ATPase-defective hsp90 blocked heme insertion into apo-iNOS by 90 and 75%, respectively. The ATPase activity of hsp90 was not required for complex formation with iNOS but was essential for heme insertion to occur. We conclude that hsp90 plays a primary role in maturation of iNOS protein by interacting with the apoenzyme in cells and then driving heme insertion in an ATP-dependent manner.
The FASEB Journal 02/2011; 25(6):2049-60. · 5.71 Impact Factor
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ABSTRACT: Although the insertion of heme into proteins enables their function in bioenergetics, metabolism, and signaling, the mechanisms and regulation of this process are not fully understood. We developed a means to study cellular heme insertion into apo-protein targets over a 3-h period and then investigated how nitric oxide (NO) released from a chemical donor (NOC-18) might influence heme (protoporphyrin IX) insertion into seven targets that present a range of protein structures, heme ligation states, and functions (three NO synthases, two cytochrome P450's, catalase, and hemoglobin). NO blocked cellular heme insertion into all seven apo-protein targets. The inhibition occurred at relatively low (nM/min) fluxes of NO, was reversible, and did not involve changes in intracellular heme levels, activation of guanylate cyclase, or inhibition of mitochondrial ATP production. These aspects and the range of protein targets suggest that NO can act as a global inhibitor of heme insertion, possibly by inhibiting a common step in the process.
Free radical biology & medicine 03/2010; 48(11):1548-58. · 5.42 Impact Factor
