Influence of the protein matrix on intramolecular histidine ligation in ferric and ferrous hexacoordinate hemoglobins

Abstract

Present in most organisms, hexacoordinate hemoglobins (hxHbs) are proteins that have evolved the capacity for reversible bis-histidyl heme coordination. The heme prosthetic group enables diverse protein functionality, such as electron transfer, redox reactions, ligand transport, and enzymatic catalysis. The reactivity of heme is greatly effected by the coordination and noncovalent chemical environment imposed by its connate protein. Of considerable interest is how the hxHb globin fold achieves reversible intramolecular coordination while still enabling high-affinity binding of oxygen, nitric oxide, and other small ligands. Here we explore this question by examining the role of the protein matrix on coordination behavior in a group of hxHbs from animals, plants, and bacteria, including human neuroglobin and cytoglobin, a nonsymbiotic hemoglobin from rice, and a truncated hemoglobin from the cyanobacterium Synechocystis. This is done with a set of experiments measuring the reduction potentials of each wild-type hxHb and its corresponding mutant protein where the reversibly bound histidine (the distal His) has been replaced with a noncoordinating side chain. These reduction potentials, coupled with studies of the mutant proteins saturated with exogenous imidazole, enable us to assess the effects of the protein matrices on histidine coordination. Our results show significant variation among the hxHbs, demonstrating flexibility in the globin moiety's ability to regulate reversible coordination. This regulation is particularly evident in the plant nonsymbiotic hemoglobins, where ferric state histidine coordination affinity is substantially lowered by the protein matrix.

A cis-Proline in α-Hemoglobin Stabilizing Protein Directs the Structural Reorganization of α-Hemoglobin

“…␣Hb from human blood was purified as described previously (3). Isotopic labeling of ASHP was achieved by expression in shaker flasks with 15 NH 4 Cl and 13 C-glucose as sole nitrogen and carbon sources (26). Met-␣Hb was prepared from oxy-␣Hb by oxidation with 5 molar eq of K 3 Fe(CN) 6 and filtered over Sephadex G-25 (Amersham Biosciences).…”

“…Side chain 13 C assignments were made from the CC(CO)NH experiment (28). The ratio of the cis/trans Xaa 29 -Pro 30 peptide bond isomers was determined from 15 N-HSQC peak volumes. X-ray Crystallography-A complex between oxy-␣Hb and AHSP residues 1-91 (AHSP-(1-91)) was produced as described (5) and oxidized by 4 molar eq of K 3 Fe(CN) 6 .…”

“…2C, lane 12), confirming that the stimulating effect of the D29W mutation relies upon the presence of Pro 30 . To investigate the structural consequences of these mutations, we assigned backbone 15 N, H N , and 13 C ␣ resonances for the D29A, D29F, D29W, L31A, and L31W mutants. All mutants displayed evidence of distinct cis/trans isomers (supplemental Fig.…”

“…To determine whether AHSP undergoes any conformation changes concomitant with ␣Hb conversion, we incubated unlabeled oxy-␣Hb with 15 N-labeled AHSP-(1-90) at 30°C and recorded 15 N-HSQC spectra at 22-min intervals for 18 h. The evolution of the final bishis-␣Hb⅐AHSP complex was confirmed by UV-visible spectroscopy. Only small changes in the 15 N-HSQC spectra were observed in this experiment (Fig.…”

“…The formation of this complex inhibits ␣Hb peroxidase activity and heme loss (2). Bishistidyl heme coordination is becoming increasingly recognized as a feature of numerous vertebrate and non-vertebrate globins (14) and has been shown previously to confer a relative stabilization of the Fe 3ϩ over the Fe 2ϩ oxidation state (15)(16)(17). Although bis-histidyl heme coordination has previously been detected in solutions of met-Hb, formed through spontaneous autoxidation of Hb (18 -21), the bishis-␣Hb⅐AHSP complex provides the first evidence that the bis-histidyl heme may play a positive functional role in Hb biochemistry by inhibiting the production of harmful reactive oxygen species.…”