|Gopalasubramniam, S - UAEM-MEXICO|
|Kovacs, Frank - UNIVERSITY OF NEBRASKA|
|Violante-Mota, F - UAEM-MEXICO|
|Twig, Paul - UNIVERSITY OF NEBRASKA|
|Arredondo-Peter, Raul - UAEM-MEXICO|
Submitted to: Proteins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 15, 2007
Publication Date: July 1, 2008
Citation: Gopalasubramniam, S., Kovacs, F., Violante-Mota, F., Twig, P., Arredondo-Peter, R., Sarath, G. 2008. Cloning and Characterization of a Caesalpinoid (Chamaecrista fasciculate) Hemoglobin: The Structural Transition from a Non-Symbiotic Hemoglobin to a Leghemoglobin. Proteins 72: 252-260. Interpretive Summary: All plants contain non symbiotic hemoglobins (nsHb), which can bind oxygen, but are unable to transport it. In contrast legumes produce symbiotic hemoglobins (symHb), in root nodules that can both bind and transport oxygen. The differences in the two types of hemoglobins reside in their protein structure and in the biophysical properties of the heme-pocket, where oxygen is reversibly bound. During plant evolution, the nsHb gave rise to symHb. The process of how this occurred is not known and the discovery of symHb proteins with strong relatedness to nsHb protein can help in elucidating this important transition. In this paper we have modeled the symHb from a primitive native prairie legume (partridge pea; pp), and have shown that the ppsymHb is a potential evolutionary intermediate in the pathway leading from nsHb to symHb.
Technical Abstract: Non-symbiotic hemoglobins (nsHbs) and leghemoglobins (Lbs) are plant proteins that can reversibly bind O2 and other ligands. The nsHbs are hexacoordinate and appear to modulate cellular concentrations of NO and maintain energy levels under hypoxic conditions. The Lbs are pentacoordinate and facilitate the diffusion of O2 to symbiotic bacteroids within legume root nodules. Multiple lines of evidence suggest that all plant Hbs evolved from a common ancestor and that Lbs originated from nsHbs. However, little is known about the structural intermediates that ocurred during the evolution of pentacoordinate Lbs from hexacoordinate nsHbs. We have cloned and characterized a Hb (ppHb) from the root nodules of the ancient caesalpinoid legume Chamaecrista fasciculata. Protein sequence, modeling data and spectral analysis indicated that the properties of ppHb are intermediate between that of nsHb and Lb, suggesting that ppHb resembles a putative ancestral Lb. Predicted structural changes that appear to have occurred during the nsHb to Lb transition were a compaction of the CD-loop and decreased mobility of the distal His inhibiting its ability to coordinate directly with the heme-Fe, leading to a pentacoordinate protein. Other predicted changes include shortening of the N- and C-termini, compaction of the protein into a globular structure, disappearance of positive charges outside the heme pocket and appearance of negative charges in an area located between the N- and C-termini. A major consequence for some of these changes appears to be the decrease in O2-affinity of ancestral nsHb, which resulted in the origin of the symbiotic function of Lbs.