Cloning and characterization of a caesalpinoid (Chamaecrista fasciculata) hemoglobin: The structural transition from a nonsymbiotic hemoglobin to a leghemoglobin

Abstract

Nonsymbiotic hemoglobins (nsHbs) and leghemoglobins (Lbs) are plant proteins that can reversibly bind O(2) 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 O(2) 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 occurred 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 O(2)-affinity of ancestral nsHb, which resulted in the origin of the symbiotic function of Lbs.

Function and evolution of nodulation genes in legumes

“…Therefore, expression of Lb genes is also likely to be regulated by cytokinin, which plays important roles in RN symbiosis [100]. It is noteworthy that symbiotic Hbs in the actinorhiza plant Casuarina glauca and the caesalpinioid legume Chamaecrista fasciculata (which may have evolved nodulation independently from model papilionoid legumes) show functionally intermediate properties between Lbs and class-2 nsHbs [101,102], indicating Lbs evolved before legume diversification. The symbiotic Hb in non-legume Parasponia andersonii, which establishes RN symbiosis with Bradyrhizobium spp., has a root-nodule-specific promoter and has evolved from class-1 nsHbs [103,104], so that recruitment of nsHbs to symbiotic Hbs has occurred multiple times.…”

Venturing Beyond Beans and Peas: What Can We Learn from Chamaecrista?

“…Hirsch et al (2009) propose that Rhizobium infection threads in the nodule enter secondarily through crack entry since rhizobial infection threads abort in root hairs. Within nodules, a hemoglobin with reduced O 2 affinity has been characterized that is intermediate between nonsymbiotic hemoglobins and leghemoglobin, resembling a putative ancestral leghemoglobin (Gopalasubramaniam et al, 2008). Rhizobial strains selected for C. fasciulata were collected by the late Peter Graham and will become available from the U.S. Department of Agriculture Agricultural Research Service National Rhizobium Germplasm Resource Collection curated by Peter van Berkum.…”

Rice (Oryza) hemoglobins

“…94 mya. Likewise, the structural analysis of primitive nsHbs and Lbs revealed that changes during the evolution of nsHbs to Lbs were a hexacoordinate to pentacoordinate transition at the heme prosthetic group, a length decrease at the CD-loop and N- and C-terminal regions, and a compaction of the protein into a globular structure 47 , 125 .…”