BBMB Research Seminars

April 5

Benoit Smagghe
Department of Biochemistry, Biophysics & Molecular Biology
Iowa State University
"Biophysical and Biochemical Characterization of Hexacoordinate Hemoglobins"

Abstract:

Within the hemoglobin (Hb) superfamily, an unusual group called hexacoordinate hemoglobin (hxHbs) whose members are found in all living organisms is the subject of exciting studies since their discovery. They are named “hexacoordinate” because of reversible coordination of the sixth coordination site of the heme iron by a histidine side chain located in the heme distal pocket. Interestingly, hxHbs share high degree of similarity in their primary sequence indicating that they might share a common role and that endogenous coordination might be an important part of their not yet characterized physiological function.  The characterization of their ligand binding mechanism and their biochemical activity are the first steps toward the understanding of their function.

Using flash photolysis and rapid mixing methods, we measured the CO binding to human, plant and bacterial hxHbs. The results obtained using our new model demonstrate that hexacoordination regulates, at different degree, the affinity constants for ligand binding. Plant hxHbs contain several conserved amino acids, in particular a Phe at position "B10" (PheB10) that is near the reversibly coordinated distal HisE7 and a Cys at position “E16” (CysE16) that could be involved in nitric oxide scavenging. We have investigated the effects of PheB10 and CysE16 mutations on kinetic and equilibrium constants for hexacoordination and exogenous ligand binding in the ferrous and ferric oxidation states.  Our analyses reveal that PheB10 and CysE16 are key regulatory element in hexacoordination and that CysE16 is not crucial for the NO dioxygenation.

Phylogenetically, plant hxHbs are grouped into two different classes: class 1 and class 2. No reports comparing the oxygen affinity of plant hxHbs from different class are available. Therefore, we have determined the components necessary to determine accurately the oxygen affinity for class 1 and 2 hxHbs. Our results are the first evidence that each class of plant hxHbs might have different functions in their respective environment.

Despite considerable attention to these hxHbs, no clear physiological role(s) has been assigned to them in any species. One popular and relevant hypothesis for their function is NO dioxygenation. Therefore, we have tested different hxHbs for their abilities to scavenge NO. Based on in vitro analysis, we conclude that there is no characteristic that distinguishes hxHbs from other Hbs that could support their predisposal toward a role in NO scavenging. However, when comparing their ability to substitute for flavoHb (a known NO scavenger) in E. coli, Synechocystis hemoglobin is the only one to confer protection from NO in vivo while the other hxHbs and myoglobin fail in this function.

In conclusion, the biophysical and biochemical characterization of several hxHbs presented here is an important contribution towards the understanding of hxHbs physiological function.