The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme

Abstract

Sperm whale myoglobin (Mb) and soybean leghemoglobin (Lba) are two small, monomeric hemoglobins that share a common globin fold but differ widely in many other aspects. Lba has a much higher affinity for most ligands, and the two proteins use different distal and proximal heme pocket regulatory mechanisms to control ligand binding. Removal of the constraint provided by covalent attachment of the proximal histidine to the F-helices of these proteins decreases oxygen affinity in Lba and increases oxygen affinity in Mb, mainly because of changes in oxygen dissociation rate constants. Hence, Mb and Lba use covalent constraints in opposite ways to regulate ligand binding. Swapping the F-helices of the two proteins brings about similar effects, highlighting the importance of this helix in proximal heme pocket regulation of ligand binding. The F7 residue in Mb is capable of weaving a hydrogen-bonding network that holds the proximal histidine in a fixed orientation. On the contrary, the F7 residue in Lba lacks this property and allows the proximal histidine to assume a conformation favorable for higher ligand binding affinity. Geminate recombination studies indicate that heme iron reactivity on picosecond timescales is not the dominant cause for the effects observed in each mutation. Results also indicate that in Lba the proximal and distal pocket mutations probably influence ligand binding independently. These results are discussed in the context of current hypotheses for proximal heme pocket structure and function.

Effects of Distal Pocket Mutations on the Geminate Recombination of NO with Leghemoglobin on the Picosecond Time Scale

“…These two observations can be reconciled if it is postulated that the mutation introduces an opening in the heme pocket, which, while were selected, as discussed in detail elsewhere, because they are complimentary substitutions resulting in an Lba-like proximal pocket in Mb (S92V) and an Mb-like proximal pocket in Lba (V91S). 6 (B) For the two panels (a) and (b), the top portion represents the kinetic traces (400 ps full time scale) and the bottom the corresponding distributions for wtLba and its distal mutants obtained from MEM analysis. permitting the ligand to stray further from the Fe (thus providing slow geminate rebinding), also permits easy reentry for ligands that have already exited (fast bimolecular rebinding).…”

The Crystal Structure of Synechocystis Hemoglobin with a Covalent Heme Linkage

“…In general, it is thought that staggering of the azimuthal angle of the proximal histidine away from the heme pyrrole nitrogens increases ligand affinity by allowing the iron to move into the plane of the porphyrin ring (56,57). This holds true for SynHb, RiceHb, and Lba, which are all staggered and which all have relatively high oxygen affinities compared with Mb (17,22,58).…”

The Complex of Apomyoglobin with the Fluorescent Dye Coumarin 153¶

“…Key histidine residues in the heme pocket are also shown. Residue Val68 (one of the residues in contact with C153 in the hydrophobic heme pocket with small nuclear polarizability) is used in one of our mutations (47,48).…”

“…First, coumarins in general, and C153 in particular, are wellcharacterized and widely used chromophores for solvation dynamics studies (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46). Second, we can produce a broad range of mutant proteins (47,48) in which one or several amino acids are strategically replaced, so as to test our theoretical model. Third, the structures of many myoglobins and their mutants have been determined to high resolution (49,50).…”