Two hexacoordinate hemoglobins (hxHbs) have very recently been identified in humans. Neuroglobin (neuroHb) was the first human hxHb discovered, and is localized in the brain. We have identified a second human hxHb that is currently referred to as histoglobin (histoHb), and is found in nearly all tissues. HxHbs are a class of proteins found in a wide array of organisms including animals, plants, and photosynthetic microorganisms. The nomenclature """"""""hxHb"""""""" denotes a hemoglobin with intramolecular coordination of the ligand binding site similar to cytochrome b5. In spite of this obstruction (cytochrom b5 does not bind gaseous ligands), hxHbs bind ligands reversibly and with apparently high affinities. NeuroHb is thought to play a role in cell survival during hypoxia, but no physiological roles have yet been established with confidence for these proteins. ? ? Our long range goal is to deduce the physiological role(s) of human hxHbs, and to understand how their function is dictated by protein structure. The objective of this proposal is to measure the affinities of human hxHbs for ligands including 02 and nitric oxide (NO), to identify basic regulatory elements of these proteins, and to evaluate their capacity to scavenge NO both in vitro and in vivo. Our central hypothesis is that neuroHb and histoHb are high affinity Hbs that use reversible hexacoordination and protein conformational changes to regulate ligand binding. We further suggest that neuroHb serves to scavenge NO in neural tissues. The rationale underlying our objective is that fundamental examination of the reactions of these proteins with their biological ligands is crucial in ascertaining their potential physiological roles. ? ? Four specific aims are proposed to test our hypotheses. 1) Characterization of 02, CO, NO, and CN- binding to neuroHb. 2) Elucidation of the structure and mechanism of ligand binding for human neuroHb. 3) Examination of the NO dioxygenation activity of neuroHb, and evaluation of its physiological relevance. 4) Structural and biophysical characterization of histoHb, and its ability to function as an NO dioxygenase. ? ? The study of both hemoglobins and NO have been important topics in medical research for some time. But a large body of recent work has made it clear that we are only beginning to realize the physiological significance of their interactions with one another. Our work is critical as hxHbs are proving to be biologically ubiquitous molecules, and it is imperative that we assess their potential involvement in NO homeostasis in humans. Additionally, our experiments with neuroHb and histoHb will develop general principles that will be applicable to all hxHb. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM065948-01A1
Application #
6612200
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Cassatt, James
Project Start
2003-07-01
Project End
2007-06-30
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
1
Fiscal Year
2003
Total Cost
$230,959
Indirect Cost
Name
Iowa State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
005309844
City
Ames
State
IA
Country
United States
Zip Code
50011
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Smagghe, Benoit J; Trent 3rd, James T; Hargrove, Mark S (2008) NO dioxygenase activity in hemoglobins is ubiquitous in vitro, but limited by reduction in vivo. PLoS One 3:e2039
Halder, Puspita; Trent 3rd, James T; Hargrove, Mark S (2007) Influence of the protein matrix on intramolecular histidine ligation in ferric and ferrous hexacoordinate hemoglobins. Proteins 66:172-82
Hoy, Julie A; Robinson, Howard; Trent 3rd, James T et al. (2007) Plant hemoglobins: a molecular fossil record for the evolution of oxygen transport. J Mol Biol 371:168-79
Hoy, Julie A; Smagghe, Benoit J; Halder, Puspita et al. (2007) Covalent heme attachment in Synechocystis hemoglobin is required to prevent ferrous heme dissociation. Protein Sci 16:250-60
Smagghe, Benoit J; Sarath, Gautam; Ross, Emily et al. (2006) Slow ligand binding kinetics dominate ferrous hexacoordinate hemoglobin reactivities and reveal differences between plants and other species. Biochemistry 45:561-70
Smagghe, Benoit J; Kundu, Suman; Hoy, Julie A et al. (2006) Role of phenylalanine B10 in plant nonsymbiotic hemoglobins. Biochemistry 45:9735-45
Weiland, Theodore R; Kundu, Suman; Trent 3rd, James T et al. (2004) Bis-histidyl hexacoordination in hemoglobins facilitates heme reduction kinetics. J Am Chem Soc 126:11930-5