Nuclear Magnetic Resonance Investigation of the Interaction of Heme Binding Proteins with SnIVprotoporphyrin IX and Heme: Structure and Conformational Changes of Myoglobin and Hemopexin

• Main Author: Deeb, Ruba Saba
• Format: Others
• Published: PDXScholar 1993
• Subjects: Nuclear magnetic resonance spectroscopy; Heme; Hemoproteins
• Online Access: https://pdxscholar.library.pdx.edu/open_access_etds/1368; https://pdxscholar.library.pdx.edu/cgi/viewcontent.cgi?article=2367&context=open_access_etds

Tin protoporphyrin IX (SnPP) is currently under investigation for the treatment of hyperbilirubinemia. The study of the complex between SnPP and equine myoglobin (EqMb) by ¹H and ¹¹⁹Sn nuclear magnetic resonance spectroscopy (NMR) can be viewed as a general model for SnPP interaction with hemoproteins. The complex formed from the equilibrium mixture of SnPP and EqMb, SnPP•EqMb, was found to have essentially the same porphyrin-binding pocket as EqMbCO and SwMbCO, including the same porphyrin orientation in the major form of the two species. ¹¹⁹Sn NMR spectroscopy was used to demonstrate that the proximal His(93)F8-metal coordination is likely to be intact in SnPP•EqMb. Minor shifts in the side chain positions of some of the residues were observed, possibly reflecting the presence of water in the sixth coordination site. SnPP•EqMb appears to be stable; it persists at room temperature for weeks and exhibits very slow exchange rates (²Hfor ¹H) for a large number of amide protons in the pH range 7-9. Events during the reconstitution of apomyoglobin (apoMb) with SnPP were probed. Thus interactions between tin(IV)protoporphyrin IX (SnPP) and equine apoMb, and between tin(IV) protoporphyrin IX dimers (SnPP)₂ and apoMb were observed by ¹H NMR and optical spectroscopic techniques. The products and intermediates observed in this situation were related to the equilibrium structure of SnPP•EqMb. Reactions of apoEqMb with SnPP and (SnPP)₂ produce different intermediates, although the final product, SnPP•EqMb, is the same for each. An intermediate observed for the reaction of SnPP with apoEqMb at pH 10 is in exchange with free SnPP, with the observed rate constant Koff ~ 1 sˉ¹; meso-proton resonances were assigned for this intermediate by correlation to SnPP resonances via chemical exchange. The intermediate observed for the reaction of (SnPP)₂ with apoEqMb at neutral pH produces another species which may be the alternate porphyrin-insertion isomer arising from a 180° rotation about the α,γ-meso axis of the porphyrin. Although optical absorbance spectroscopy of the Soret region shows evidence for the reaction of SnPP and (SnPP)₂ with apoMb, only in combination with ¹H NMR are the various processes assigned. T his study of the complex SnPP•EqMb facilitated the investigation of the more complex heme binding protein, hemopexin (Hx). Proton NMR spectroscopy is reported for the first time for the hemin complex of hemopexin, a serum protein that binds heme exceptionally tightly. Hx from cow, rat, rabbit, and human was isolated, and data for the protein were reported. Heme-bound Hx has spectral characteristics for being low-spin, paramagnetic. Deuterium isotope labels reveal the positions for the heme 1-, 3-, and 8-methyls; the 5-methyl lies in the -5 to 12 ppm region. Furthermore, two-dimensional nuclear Overhauser effect spectroscopy was used to locate other heme periphery protons, including those from the 2-vinyl and the 7-propionate. Upfield resonances are identified that are very strongly relaxed, and so are assigned to protons on the axial ligands. The information reported here contributes to the understanding of Hx as an antioxidant at the cellular level.