All plants contain an unusual class of hemoglobins that display bis-histidyl coordination yet are able to bind exogenous ligands such as oxygen. Structurally homologous hexacoordinate hemoglobins (hxHbs) are also found in animals (neuroglobin and cytoglobin) and some cyanobacteria, where they are thought to play a role in free radical scavenging or ligand sensing. The plant hxHbs can be distinguished from the others because they are only weakly hexcacoordinate in the ferrous state, yet no structural mechanism for regulating hexacoordination has been articulated to account for this behavior. Plant hxHbs contain a conserved Phe at position B10 (Phe(B10)), which is near the reversibly coordinated distal His(E7). We have investigated the effects of Phe(B10) mutation on kinetic and equilibrium constants for hexacoordination and exogenous ligand binding in the ferrous and ferric oxidation states. Kinetic and equilibrium constants for hexacoordination and ligand binding along with CO-FTIR spectroscopy, midpoint reduction potentials, and the crystal structures of two key mutant proteins (F40W and F40L) reveal that Phe(B10) is an important regulatory element in hexacoordination. We show that Phe at this position is the only amino acid that facilitates stable oxygen binding to the ferrous Hb and the only one that promotes ligand binding in the ferric oxidation states. This work presents a structural mechanism for regulating reversible intramolecular coordination in plant hxHbs.
Detailed comparisons of the carbon monoxide FTIR spectra and ligand-binding properties of
a library of E7, E11, and B10 mutants indicate significant differences in the role of electrostatic interactions
in the distal pockets of wild-type sperm whale myoglobin and soybean leghemoglobin. In myoglobin,
strong hydrogen bonds from several closely related conformations of the distal histidine (HisE7) side chain
preferentially stabilize bound oxygen. In leghemoglobin, the imidazole side chain of HisE7 is confined to
a single conformation, which only weakly hydrogen bonds to bound ligands. The phenol side chain of
TyrB10 appears to “fix” the position of HisE7, probably by donating a hydrogen bond to the Nδ atom of
the imidazole side chain. The proximal pocket of leghemoglobin is designed to favor strong coordination
bonds between the heme iron and axial ligands. Thus, high oxygen affinity in leghemoglobin is established
by a favorable staggered geometry of the proximal histidine. The interaction between HisE7 and TyrB10
prevents overstabilization of bound oxygen. If hydrogen bonding from HisE7 were as strong as it is in
mammalian myoglobin, the resultant ultrahigh affinity of leghemoglobin would prevent oxygen transport
in root nodules.
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