Nitrosylation of rabbit ferrous heme-hemopexin.
Hemopexin (HPX) serves as a trap for toxic plasma heme, ensuring its complete clearance by transportation to the liver. Moreover, HPX-heme has been postulated to play a key role in the homeostasis of nitric oxide (NO). Here, the thermodynamics for NO binding to rabbit ferrous HPX-heme as well as the EPR and optical absorption spectroscopic properties of rabbit ferrous nitrosylated HPX-heme (HPX-heme-NO) are reported. The value of the dissociation equilibrium constant for NO binding to rabbit ferrous HPX-heme (i.e., H) is (1.4+/-0.2)x10(-7) M, at pH 7.0 and 10.0 degrees C; the value of H is unaffected by sodium chloride. At pH 7.0, rabbit ferrous HPX-heme-NO is a six-coordinate heme-iron species, characterized by an X-band EPR spectrum with an axial geometry and by epsilon=146 mM(-1) cm(-1) at 419 nm. At pH 4.0, rabbit ferrous HPX-heme-NO is a five-coordinate heme-iron species, characterized by an X-band EPR spectrum with three-line splitting centered at 334 mT and by epsilon=74 mM(-1) cm(-1) at 387 nm. The p K(a) value of the reversible pH-induced six- to five-coordinate spectroscopic transition is 4.8+/-0.1 in the absence of sodium chloride and 4.3+/-0.1 in the presence of 1.5x10(-1) M sodium chloride. This result is in agreement with the effect of sodium chloride on rabbit HPX-heme stability. The present data have been analyzed in parallel with those of a related heme model compound and heme-protein systems.
Fasano
Mauro
M
Department of Structural and Functional Biology, University of Insubria, Via Alberto da Giussano 12, 21052 Busto Arsizio (VA), Italy.
Bocedi
Alessio
A
Mattu
Marco
M
Coletta
Massimo
M
Ascenzi
Paolo
P
D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006466:Hemopexin / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009566:Nitrates / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013816:Thermodynamics
D005296:Ferrous Compounds; D009566:Nitrates; D009569:Nitric Oxide; D006418:Heme; D006466:Hemopexin