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

Authors

Sabarinathan K. Gopalasubramaniam, Laboratorio de Biofisica y Biologia Molecular, Facultad de Ciencias, Universidad Autonoma del Estado de Morelos, Morelos, Mexico
Frank A. Kovacs, University of Nebraska-KearneyFollow
Fernando Violante-Mota, Laboratorio de Biofisica y Biologia Molecular, Facultad de Ciencias, Universidad Autonoma del Estado de Morelos, Morelos, Mexico
Paul Twigg, University of Nebraska-KearneyFollow
Raul Arrendondo-Peter, Laboratorio de Biofisica y Biologia Molecular, Facultad de Ciencias, Universidad Autonoma del Estado de Morelos, Morelos, Mexico
Gautam Sarath, University of Nebraska-LincolnFollow

Document Type

Article

Date of this Version

5-6-2008

Comments

Published in Proteins 2008; 72:252–260.

Abstract

Nonsymbiotic 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 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 CDloop 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.