EXPRESSION OF RECOMBINANT MONOMER HEMOGLOBINS (COMPONENT-IV) FROM THEMARINE ANNELID GLYCERA-DIBRANCHIATA - EVIDENCE FOR PRIMARY SEQUENCE POSITIONAL REGULATION OF HEME ROTATIONAL DISORDER

A description of the efficient high-level expression of the monomer he moglobin (GMG4) from Glycera dibranchiata is presented. The cDNA descr ibed by Simons and Satterlee [Simons, P. C., and Satterlee, J. D. (198 9) Biochemistry 28, 8525-8530] was subcloned into an expression system , and conditions were found that led to the production df large amount s of soluble apoprotein (rec-gmg), These conditions included lowering the temperature during the induction period and growth in a rich mediu m with a higher ionic strength. Characterization of this reconstituted recombinant protein showed that it was not identical to the native GM H4 protein. Both UV-visible and H-1 NMR data indicated differences wit hin the holoprotein (rec-gmh) heme pocket compared to the native prote in, the major difference being that two nonidentical heme orientations are significantly populated in rec-gmh. This phenomenon has been seen previously in other heme proteins, where these heme orientational iso mers are described by a 180-deg rotation about the heme alpha-gamma me sp axis. This work prompted the production of a complete chemical sequ ence for the native GMH4 [Alam S. L., Satterlee, J. D., and Edmonds, C . G: (1994) J. Protein Chem.13, 151-164], which showed that the expres sed rec-gmg protein differed at three primary sequence positions (41, 95, and 123) from the native component IV globin (GMG4). Subsequently, we have produced the triple-revertant mutations required to express t he recombinant wild-type protein (recGMG4). The physical characteristi cs of the active site in the holoprotein (recGMH4) are identical to th ose of the native protein. In addition, we have evidence, based on the model of GMH4, that position 41 may be crucial in determining the ori entation of the heme within the active site.