Introduction of a covalent histidine–heme linkage in a hemoglobin: A promising tool for heme protein engineering

• Control of both heme reactivity and affinity in designed proteins is a challenge.
• Addition of a histidine to a heme vinyl is proposed as a tool to prevent heme loss.
• Proof of concept is offered with two histidine variants of a Chlamydomonas Hb.
• NMR spectroscopy is essential to the characterization of the products.

The hemoglobins of the cyanobacteria Synechococcus and Synechocystis (GlbNs) are capable of spontaneous and irreversible attachment of the b heme to the protein matrix. The reaction, which saturates the heme 2-vinyl by addition of a histidine residue, is reproduced in vitro by preparing the recombinant apoprotein, adding ferric heme, and reducing the iron to the ferrous state. Spontaneous covalent attachment of the heme is potentially useful for protein engineering purposes. Thus, to explore whether the histidine–heme linkage can serve in such applications, we attempted to introduce it in a test protein. We selected as our target the heme domain of Chlamydomonas eugametos LI637 (CtrHb), a eukaryotic globin that exhibits less than 50% sequence identity with the cyanobacterial GlbNs. We chose two positions, 75 in the FG corner and 111 in the H helix, to situate a histidine near a vinyl group. We characterized the proteins with gel electrophoresis, absorbance spectroscopy, and NMR analysis. Both T111H and L75H CtrHbs reacted upon reduction of the ferric starting material containing cyanide as the distal ligand to the iron. With L75H CtrHb, nearly complete (> 90%) crosslinking was observed to the 4-vinyl as expected from the X-ray structure of wild-type CtrHb. Reaction of T111H CtrHb also occurred at the 4-vinyl, in a 60% yield indicating a preference for the flipped heme orientation in the starting material. The work suggests that the His–heme modification will be applicable to the design of proteins with a non-dissociable heme group.

Two histidine variants of the monomeric Chlamydomonas hemoglobin, CtrHb, were prepared to explore the feasibility of engineering a heme-histidine covalent crosslink in arbitrary heme proteins. Product yield and homogeneity depend on steric considerations and the identification of suitable reductive conditions.Figure optionsDownload as PowerPoint slide