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

Abstract

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.

Covalent attachment of the heme to Synechococcus hemoglobin alters its reactivity toward nitric oxide

“…Specifically, the GlbN PTM consists in the saturation of the 2-vinyl substituent, covalently attaching the heme to His117 Nε2 [9] (Figure S1B). The PTM is facile in that it can be engineered to occur at the 4-vinyl (Leu79His/His117Ala variant of Synechocystis GlbN and Leu75His variant of Chlamydomonas eugametos LI637 CtrHb) and even both the 2- and 4-vinyls (Leu79His variant of Synechocystis GlbN) [10, 11]. Many cyanobacterial TrHb1s have strong sequence identity around a highly conserved His117 and are suspected to undergo the PTM as well.…”

How a novel tyrosine–heme cross-link fine-tunes the structure and functions of heme proteins: a direct comparitive study of L29H/F43Y myoglobin

“…PCC 6803 25 and Synechococcus sp. 27 The cross-link can also be introduced in other globins, such as in Mb by introduction of a non-coordinating histidine close to the heme 2-vinyl group (I107H mutation), whereas no structural information on I107H Mb has been obtained yet. 27 The cross-link can also be introduced in other globins, such as in Mb by introduction of a non-coordinating histidine close to the heme 2-vinyl group (I107H mutation), whereas no structural information on I107H Mb has been obtained yet.…”

The broad diversity of heme-protein cross-links: An overview

“…Recently, Lecomte and co-workers [71] showed the application in a eukaryotic globin, Chlamydomonas eugametos LI637 (CtrHb), which has less than 50% sequence identity with the cyanobacteria Hbs. As confirmed by gel electrophoresis, absorbance spectroscopy and NMR analysis, introduction of a histidine close to heme 4-vinyl group at position 75 or 111 lead to formation of a His-heme cross-link (N90% and~60% cross-link formation in L75H CtrHb and T111H CtrHb, respectively), following the reduction of ferric protein in presence of exogenous ligand cyanide.…”

“…The inset shows the kinetic trace at 556 nm (Adapted with permission from ref. [71]. Copyright (2014) Elsevier).…”

“…With an emerging understanding, albeit not complete, of the diverse cross-links in natural heme proteins, most of these cross-links have been successfully recreated in natural or de novo proteins, such as Cys-heme [24,26,27,33,[41][42][43][44][45], Met-heme [53], His-heme [70][71][72], Trp/Tyr-heme [78] and Glu/Asp-heme [82,106,[108][109][110][111] cross-links. These achievements, in turn, have enhanced our knowledge of how heme-protein cross-link regulates the structure and function of heme proteins in nature.…”