doi:10.1016/j.jmb.2007.05.029 
Copyright © 2007 Elsevier Ltd All rights reserved.
Plant Hemoglobins: A Molecular Fossil Record for the Evolution of Oxygen Transport
Julie A. Hoy1, Howard Robinson2, James T. Trent III1, Smita Kakar1, Benoit J. Smagghe1 and Mark S. Hargrove1, 
, 
1Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
2Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA
Received 2 March 2007;
revised 7 May 2007;
accepted 9 May 2007.
Edited by R. Huber.
Available online 18 May 2007.
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Abstract
The evolution of oxygen transport hemoglobins occurred on at least two independent occasions. The earliest event led to myoglobin and red blood cell hemoglobin in animals. In plants, oxygen transport “leghemoglobins” evolved much more recently. In both events, pentacoordinate heme sites capable of inert oxygen transfer evolved from hexacoordinate hemoglobins that have unrelated functions. High sequence homology between hexacoordinate and pentacoordinate hemoglobins in plants has poised them for potential structural analysis leading to a molecular understanding of this important evolutionary event. However, the lack of a plant hexacoordinate hemoglobin structure in the exogenously ligand-bound form has prevented such comparison. Here we report the crystal structure of the cyanide-bound hexacoordinate hemoglobin from barley. This presents the first opportunity to examine conformational changes in plant hexacoordinate hemoglobins upon exogenous ligand binding, and reveals structural mechanisms for stabilizing the high-energy pentacoordinate heme conformation critical to the evolution of reversible oxygen binding hemoglobins.
Keywords: hexacoordinate hemoglobin; plant hemoglobins; barley hemoglobin; oxygen transport; evolution
Abbreviations: Hb, hemoglobin; nsHb, non-symbiotic Hb; SAD, single wavelength anomalous dispersion; hxHbs, hexacoordinate Hbs
Figure 1. Evolution of plant Hbs. Two classes evolved prior to the divergence of monocots (M) and dicots (D) from the primordial (P) plant Hb. The most common O2 transporters (the leghemoglobins, or Lb) evolved from the class 2 dicot Hbs in legumes (D2). Leghemoglobins are pentacoordinate in structure (upper dotted box; one histidine side-chain bonds to the heme iron), whereas the scavenger/sensor Hbs (D2, D1, M1) are hexacoordinate (lower dotted box; two histidine residues coordinate the heme iron).
Figure 2. BarHb:CN structure, electron density, and comparison to riceHb1. (a) Divergent stereoview of the barHb:CN structure (blue) overlayed with riceHb1 (red), with the region of conformational change shown darker. Helix labels are included for clarity. (b) Electron density for barHb:CN and (c) riceHb1 shows the breakdown of density around the CD loop region in the later.
Figure 3. Comparison of plant Hb structures. The primary sequence alignment is provided for barHb, riceHb1, and soybean Lba, color-coded by conservation using the BLOSUM62 score. Above the alignment, helices are labeled according to their position in barHb, and the rmsd between barHb and riceHb1 is shown per residue. The broken line portion indicates where the riceHb1 model is uncertain due to lack of electron density.
Figure 4. Hydrogen bonds and hydrophobic networks in the CD-loop region. The hydrogen bonds (pink) and hydrophobic residues (yellow) in the CD-loop region are shown for (a) barHb:CN (blue), (b) riceHb1 (red), and (c) Lba (green) in divergent stereoview. Helices are labeled along with some familiar amino acid residues for reference.
Figure 5. Hydrogen bonds in the EF-loop region. The hydrogen bonds (pink) in the EF-loop region are shown for (a) barHb:CN (blue), (b) riceHb1 (red), and (c) Lba (green) in divergent stereoview. Helices are labeled for reference.
Figure 6. Heme pocket comparison. Phe(B10) and the distal histidine His(E7) in riceHb1 (red) and barHb:CN (blue).
Figure 7. Primary sequence differences in Lba. Amino acid variations in Lba result in an increased number of (a) hydrogen bonds (pink) and (b) hydrophobic contacts with the E-helix in Lba compared to barHb:CN. These amino acids are shown in orange, while other important residues involved in the interactions are shown in dark green. Both panels contain the heme in grey and the acetate ligand and the distal and proximal histidine residues in green. Helices are labeled for reference.
Figure 8. Ferrous ligand binding kinetics for barley Hb. Fitted rate constants from flash photolysis are plotted versus (a) [O2] and (b) [CO] and fit to a line to provide k′O2 and k′CO respectively. (c) Fitted rate constants from rapid mixing at different CO concentrations are fitted to equation (1) to extract k−H and kH.
Table 1.
Data collection and refinement statistics
a Outer shell statistics are shown in parentheses.
b Calculated using 5% of reflections.
Table 2.
Rate constants for CO and O2, and His(E7) binding
a Duff
et al.
22b Arredondo-Peter
et al.
67c Smagghe
et al.26d Hargrove
et al.
30
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