Hemopexin has two homologous domains (N- and C-terminal domains), binds 1 mole of heme per mole with high affinity (Kd < 1 pM) in a low-spin bis-histidyl complex, and acts as a transporter for the heme. Transport is accomplished via endocytosis without degradation of the protein. Factors that affect stability of the heme coordination complex and potentially heme release in vivo were examined. The effects of temperature on hemopexin, its N-terminal domain, and their respective ferri-, ferro-, and CO-ferro-heme complexes were studied using absorbance and circular dichroism (CD) spectroscopy. As monitored with second-derivative absorbance spectra, the higher order structure of apo-hemopexin unfolds with a Tm of 52 degrees C in 50 mM sodium phosphate buffer and is stabilized by 150 mM NaCl (Tm 63 degrees C). Bis-histidyl heme coordination by hemopexin, observed by Soret absorbance, is substantially weakened by reduction of ferri-heme-hemopexin (Tm 55.5 degrees C) to the ferro-heme form (Tm 48 degrees C), and NaCl stabilizes both complexes by 10-15 degrees C. CO binding to ferroheme-hemopexin restores complex stability (Tm 67 degrees C). Upon cooling, unfolded apo- and ferriheme-hemopexin extensively refold and recover substantial heme-binding activity, but the characteristic ellipticity of the native protein (UV region) and heme complex (Soret region) are not regained, indicating that altered refolded forms are produced. Lowering the pH from 7.4 to 6.5 has little effect on the stability of the apo-protein but increases the Tm of heme complexes by 5-12 degrees C. The stability of the apo-N-terminal domain (Tm 53 degrees C) is similar to that of intact hemopexin, and the ferri-, ferro-, and CO-ferro-heme complexes of the N-terminal domain have Tm values of 53 degrees C, 33 degrees C, and 75 degrees C, respectively.