Facile Heme Vinyl Posttranslational Modification in a Hemoglobin

Abstract

Iron-protoporphyrin IX, or b heme, is utilized as such by a large number of proteins and enzymes. In some cases, notably the c-type cytochromes, this group undergoes a posttranslational covalent attachment to the polypeptide chain, which adjusts the physicochemical properties of the holoprotein. The hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803 (GlbN), contrary to the archetypical hemoglobin, modifies its b heme covalently. The posttranslational modification links His117, a residue that does not coordinate the iron, to the porphyrin 2-vinyl substituent and forms a hybrid b/c heme. The reaction is an electrophilic addition that occurs spontaneously in the ferrous state of the protein. This apparently facile type of heme modification has been observed in only two cyanobacterial GlbNs. To explore the determinants of the reaction, we examined the behavior of Synechocystis GlbN variants containing a histidine at position 79, which is buried against the porphyrin 4-vinyl substituent. We found that L79H/H117A GlbN bound the heme weakly but nevertheless formed a cross-link between His79 Nε2 and the heme 4-Cα. In addition to this linkage, the single variant L79H GlbN also formed the native His117-2-Cα bond yielding an unprecedented bis-alkylated protein adduct. The ability to engineer the doubly modified protein indicates that the histidine-heme modification in GlbN is robust and could be engineered in different local environments. The rarity of the histidine linkage in natural proteins, despite the ease of reaction, is proposed to stem from multiple sources of negative selection.

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

“…Although only a single His-heme cross-link was discovered in above two cyanobacteria Hbs, Lecomte and co-workers [70] showed that a second one can be readily created in PCC 6803 by introduction of a histidine close to the heme 4-vinyl group (L79H mutation) (Fig. 6A), His79-N ε -4-vinyl-C α , where His79 has an orientation analogous to that of His117 with respect to the heme 2-vinyl group.…”

“…Since the formation of a His-heme cross-link occurs spontaneously in vitro under reducing conditions [70], construction of a His-heme cross-link is a potential strategy for design of new proteins with a non-dissociable heme cofactor. 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.…”

“…Moreover, not only single and double cross-links, but also triple and quadruple cross-links have been discovered, as in Cyt P460 [130] and HAO [134], respectively. These heme-protein cross-links are formed either under reductive conditions such as for Cys-heme (the typical thioether bond) [41], SeCys-heme [61] and His-heme [70] cross-links, or under oxidative conditions involving the formation of catalytic Compound I, such as for Met-heme (sulfonium ion bond) [53], Trp/Tyr-heme (C-C bond) [76,135] and Glu/Asp-heme (ester bond) [89] cross-links.…”

“…For example, His-heme cross-link does not alter the heme conformation of Hb [70] and Mb [72], whereas the heme plane was considerably distorted by the Glu/Asp-heme and Met-heme cross-links in MPO [46], the Cys-heme and Lys-heme crosslinks in cyt P460 [130], and the Cys-heme and Tyr-heme cross-links in HAO [133]. On the other hand, the His-heme cross-link has minimal effects on ligand binding properties of Mb [72], whereas a Tyr-heme cross-link regulates both spectral properties and catalytic functions of Mb [83].…”

Significantly Enhanced Heme Retention Ability of Myoglobin Engineered to Mimic the Third Covalent Linkage by Nonaxial Histidine to Heme (Vinyl) in Synechocystis Hemoglobin

“…Engineering the covalent linkage to heme in other globins might also allow a new strategy to enhance heme stability. Such engineering of a covalent linkage has been attempted before but only in SynHb without the third linkage (H117A) for a different purpose at a different location and not in the classical globins (29).…”