The Truncated Oxygen-avid Hemoglobin from Bacillus subtilis

Abstract

The group II truncated hemoglobin from Bacillus subtilis has been cloned, expressed, purified, and characterized. B. subtilis truncated hemoglobin is a monomeric protein endowed with an unusually high oxygen affinity (in the nanomolar range) such that the apparent thermodynamic binding constant for O 2 exceeds that for CO by 1 order of magnitude. The kinetic basis of the high oxygen affinity resides mainly in the very slow rate of ligand release. The extremely stable ferrous oxygenated adduct is resistant to oxidation, which can be achieved only with oxidant in large excess, e.g. ferricyanide in 50-fold molar excess. The three-dimensional crystal structure of the cyano-Met derivative was determined at 2.15 Å resolution. Although the overall fold resembles that of other truncated hemoglobins, the distal heme pocket displays a unique array of hydrophilic side chains in the topological positions that dominate the steric interaction with iron-bound ligands. In fact, the Tyr-B10, Thr-E7, and Gln-E11 oxygens on one side of the heme pocket and the Trp-G8 indole NE1 nitrogen on the other form a novel pattern of the "ligand-inclusive hydrogen bond network" described for mycobacterial HbO. On the proximal side, the histidine residue is in an unstrained conformation, and the iron-His bond is unusually short (1.91 Å).

Protein structure in the truncated (2/2) hemoglobin family

“…Among these, HisE7 is the only residue fully conserved in group III (2). TyrB10 and TrpG8 are directly hydrogen bonded to the ligand, closely matching the ligand binding mode found in group II Bs-2/2HbO (14). Contrary to group II 2/2HbO, group III 2/2HbPs display an invariant Phe residue at position CD1 and a hydrophobic residue at position E11.…”

“…In Bs-2/2HbO cyano-met crystal structure TyrB10 is the residue directly hydrogen bonded to the ligand. GlnE11, TrpG8, and ThrE7 complete the distal site polar cage, with GlnE11 side chain and the TrpG8 indolic nitrogen atom hydrogen bonded to the heme ligand (14). The occurrence of TyrCD1, however, is sufficient to drastically modify the hydrogen bonding distal network in Mt-2/2HbO, where TyrCD1, not TyrB10, is the residue hydrogen bonding to the heme diatomic ligand (13).…”

“…Further stabilizing polar interactions are provided in Mt-2/2HbO by TrpG8, whose indole NE1 atom is hydrogen bonded to the heme-bound ligand and to TyrCD1 OH; notably, residue E11 is Leu. Thus, the group II (and III) conserved TrpG8 residue can stabilize the heme ligand with one hydrogen bond, and may modulate the rate of ligand escape from the distal pocket (13,14). In the Mt-2/2HbO case, residue TyrB10 may be firstly involved in stabilizing the orientation of CD1 and G8 residues.…”

“…(Ss-2/2HbN) (10 -12), four group II HbOs from M. tuberculosis (Mt-2/ 2HbO) (13), from Bacillus subtilis (Bs-2/2HbO) (14), from Thermobifida fusca (15), from Geobacillus stearothermophilus (16), and one group III HbP from C. jejuni (Cj-2/ 2HbP) (17).…”

Ligand migration in the truncated hemoglobin of Mycobacterium tuberculosis

“…The high oxygen affinity displayed by most trHbs renders their role as oxygen transporters unlikely (4,9). Other functions such as terminal oxidases and oxygen sensors involved in the response to oxidative and nitrosative stress, nitric oxide (NO) detoxification, O 2 /NO chemistry, O 2 delivery under hypoxic conditions, and long-term ligand storage have been proposed (4,(11)(12)(13)(14).…”