Nitric oxide blocks cellular heme insertion into a broad range of heme proteins

Abstract

Although heme insertion into proteins enables their function in bioenergetics, metabolism, and signaling, the mechanisms and regulation of this process is not fully understood. We developed a means to study cellular heme insertion into apo-protein targets over a 3 h time 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 structure, heme ligation, and function (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 level, 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.

GAPDH regulates cellular heme insertion into inducible nitric oxide synthase

“…More recently, we showed that NO can inhibit cellular heme insertion into a broad range of mammalian proteins, including all three NOS enzymes, hemoglobin, catalase, and cytochrome P450 (17). Importantly, the mechanism of NO action did not involve changes in heme availability or effects of NO on cellular energy production or activation of soluble guanylate cyclase (17). The results implied that NO might act on an uncharacterized yet common cellular component or process to generally inhibit heme insertion into proteins.…”

“…In that study, the NO was produced naturally by iNOS in cells and was found to act somewhere near or at the point of heme insertion. More recently, we showed that NO can inhibit cellular heme insertion into a broad range of mammalian proteins, including all three NOS enzymes, hemoglobin, catalase, and cytochrome P450 (17). Importantly, the mechanism of NO action did not involve changes in heme availability or effects of NO on cellular energy production or activation of soluble guanylate cyclase (17).…”

Soluble guanylyl cyclase requires heat shock protein 90 for heme insertion during maturation of the NO-active enzyme

“…Stock solutions of novobiocin were prepared in water whereas 3-isobutyl-1-methylxanthine (IBMX) and radicicol were dissolved in DMSO. Stock solutions of hemin were freshly prepared in 0.1 N NaOH (32). cDNAs for human hsp90β and D88N-hsp90β mutant were gifts from Bill Sessa (Yale University, New Haven, CT).…”

“…RLF-6 cells were cultured in Ham F-12 K media containing L-glutamine and pyruvate, 5,000 U/L of penicillin/streptomycin and 20% (vol/vol) FBS. Heme depletion was achieved by culturing cells with 400 μM SA for 48 h before transfection or activation of sGC (30,32). RFL-6 cells expressing endogenous sGC were activated by NO donor SNAP (50 μM) or with heme-dependent (BAY 41-2272; 10 μM) or heme-independent activators (BAY 60-2770; 10 μM) from 0 to 30 min as indicated.…”

“…Given the pervasive nature of the sGChsp90 association, we wondered if hsp90 might play a similar role in enabling heme insertion into sGC during its maturation. To address this, we adopted a mammalian cell culture approach (30,32) that allowed us to follow heme insertion into transiently transfected and endogenously expressed apo-sGC. We used pharmacological hsp90 inhibitors, an ATPase-inactive hsp90 mutant, and heme-dependent or heme-independent sGC activators as tools to decipher the role of hsp90.…”