Unusually Strong H-Bonding to the Heme Ligand and Fast Geminate Recombination Dynamics of the Carbon Monoxide Complex of Bacillus subtilis Truncated Hemoglobin

Abstract

The active site of the oxygen-avid truncated hemoglobin from Bacillus subtilis has been characterized by infrared absorption and resonance Raman spectroscopies, and the dynamics of CO rebinding after photolysis has been investigated by picosecond transient absorption spectroscopy. Resonance Raman experiments on the CO bound adduct revealed the presence of two Fe-CO stretching bands at 545 and 520 cm-1, respectively. Accordingly, two C-O stretching bands at 1924 and 1888 cm-1 were observed in infrared absorption and resonance Raman measurements. The very low C-O stretching frequency at 1888 cm-1 (corresponding to the extremely high RR stretching frequency at 545 cm-1) indicates unusually strong hydrogen bonding between CO and distal residues. On the basis of a comparison with other truncated hemoglobin it is envisaged that the two CO conformers are determined by specific interactions with the TrpG8 and TyrB10 residues. Mutation of TrpG8 to Leu deeply alters the hydrogen-bonding network giving rise mainly to a CO conformer characterized by a Fe-CO stretching band at 489 cm-1 and a CO stretching band at 1958 cm-1. Picosecond laser photolysis experiments carried out on the CO bound adduct revealed dynamical processes that take place within a few nanoseconds after photolysis. Picosecond dynamics is largely dominated by CO geminate rebinding and is consistent with strong H-bonding contributions of TyrB10 and TrpG8 to ligand stabilization.

Following Ligand Migration Pathways from Picoseconds to Milliseconds in Type II Truncated Hemoglobin from Thermobifida fusca

“…The ∼ 2 ns time constant and the large amplitude (57% of the total absorbance change) of this kinetic phase suggest that the barrier for recombination is low and/or the barrier for escape is high. The geminate recombination in Tf- trHb is similar to that reported for the CO complexes of Bacillus subtilis Bs- trHb [26] and Micobacterium tubercolosis Mt- trHbO [24], [28]. Accordingly, the three proteins ( Tf -trHb, Bs -trHb and Mt -trHbO) exhibit very similar distal heme pocket architecture with highly conserved polar residues.…”

“…Recent works clearly indicated that in Tf -trHb TrpG8 and TyrCD1 are mainly involved in the stabilization of exogenous ligands, namely sulfide [17] or fluoride [21] in the ferric state, and CO in the ferrous state [22]. A similar network of interaction has also been described in Mycobacterium tuberculosis [23]–[25] and Bacillus subtilis trHbOs [26], [27]. However, information on the dynamic behavior of group II truncated hemoglobins upon CO photolysis is limited to partial observation demonstrating the presence of an efficient and fast geminate recombination process in the picosecond-nanosecond time scale [24]–[26], [28].…”

“…Accordingly, the three proteins ( Tf -trHb, Bs -trHb and Mt -trHbO) exhibit very similar distal heme pocket architecture with highly conserved polar residues. These residues are in contact with the iron-bound CO coordination shell in a common pattern defined “ligand inclusive hydrogen bond network”, representing a considerable barrier to ligand escape as demonstrated by the efficient geminate rebinding process [23], [26].…”

Structural flexibility of the heme cavity in the cold‐adapted truncated hemoglobin from the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125

“…The important role of Trp(109)G8 in ligand stabilization has been observed previously in other TrHbIIs, e.g. Bs-2/2HbO [45,47], Tf-2/2HbO [46] and Mt-2/2HbO [37,44]. No significant interactions of the CO ligand with His(58)CD1 are apparent from the simulations (data not shown).…”

Kinetic Analysis of a Globin-Coupled Histidine Kinase, AfGcHK: Effects of the Heme Iron Complex, Response Regulator, and Metal Cations on Autophosphorylation Activity

“…However, it is qualitatively similar to that of truncated bacterial hemoglobins. 37,38 Analyses of the kinetics of CO dissociation can be used to sensitively probe the heme environment, as demonstrated by experiments on genetically modified proteins. 36,38−41 This is also the case for Af GcHK ( Figure S2).…”