Chlorophyllide-substituted hemoglobin tetramers and hybrids: preparation, characterization, and energy transfer

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Abstract
Three chlorophyllide-substituted human Hb complexes were prepared: the tetrameric complex in which zinc pyrochlorophyllide .alpha. (ZnPChla) is substituted for all 4 hemes and the 2 complementary hybrids in which ZnPChla is substituted for heme in either the .alpha.- or .beta.-chains, while heme remains in the other chains. In each of there complexes, intramolecular Chl-Chl singlet energy transfer occurs. A variety of probes demonstrate that ZnPCHla-deoxyheme hybrids and the ZnPChla-Hb complexes consistently exhibit properties associated with the well-known T-state tertiary and quaternary structure of deoxyHb itself. Using the known crystal structure of human deoxyHb, the steady-state fluoresence anisotropy of these complexes was analyzed within the framework of the Foerster energy-transfer theory. The result is the determination of the orientation of the Qy transition dipole moment of ZnPChla. NMR data for the hybrids offer insight into specific tertiary structural changes in the heme pocket surrounding the diamagnetic ZnPChla, which accompany changes in the ligation state of the heme on the opposite chain.