Chemical reactivity of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 hemoglobins: covalent heme attachment and bishistidine coordination

Abstract

In the absence of an exogenous ligand, the hemoglobins from the cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 coordinate the heme group with two axial histidines (His46 and His70). These globins also form a covalent linkage between the heme 2-vinyl substituent and His117. The in vitro mechanism of heme attachment to His117 was examined with a combination of site-directed mutagenesis, NMR spectroscopy, and optical spectroscopy. The results supported an electrophilic addition with vinyl protonation being the rate-determining step. Replacement of His117 with a cysteine demonstrated that the reaction could occur with an alternative nucleophile. His46 (distal histidine) was implicated in the specificity of the reaction for the 2-vinyl group as well as protection of the protein from oxidative damage caused by exposure to exogenous H2O2.

Facile Heme Vinyl Posttranslational Modification in a Hemoglobin

“…As a rapid test for heme modifications, the hemochromogen assay 25 was performed as previously reported. 20 DT-reacted samples were typically reduced for 1−2 h to allow for complete conversion prior to the samples being assayed. The results were interpreted by comparison with published spectra (α and β band maxima).…”

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

“…6B), respectively, as well as NMR structure for PCC 7002 (PDB ID: 2KSC [67]). The combination of site-directed mutagenesis, NMR and optical spectroscopic studies provided evidence that the His-heme cross-link in reduced cyanobacteria Hbs was formed by an electrophilic addition with a reduction-driven mechanism (Scheme 4) [68]. Moreover, the His-heme cross-link was found to be self-amplified with the intrinsic ability of cyanobacteria Hbs to perform interprotein electron exchange [69].…”

“…Moreover, the His-heme cross-link was found to be self-amplified with the intrinsic ability of cyanobacteria Hbs to perform interprotein electron exchange [69]. It was shown that introduction of a cysteine at position 117 (H117C mutation) of PCC 6803 can result in formation of a thioether bond with the heme 2-vinyl group [68], suggesting that this position is suitable for both His-heme and Cys-heme cross-links. Despite the fact, cyanobacteria Hbs prefer the His-heme cross-link, likely due to its advantages such as less interference under high oxidative and nitrosative stress conditions that many cyanobacteria encounter [67].…”

“…For Scheme 4. Proposed reduction-driven mechanism for the His-heme cross-link [68]. example, biochemical studies of EPO showed that Asp93 in the light polypeptide chain and Glu241 in the heavy polypeptide chain form two ester bonds with the heme, although the link of Asp93 to the light chain is only partly formed in vivo [85].…”

“…Cyt b of cyt b 6 f; N57C cyt b 5 ; N57C/S71C cyt b 5 ; R98C/Y101C cyt b 562 ; S160C APX; H117C PCC 6803; Cyt P460; HAO Refs [21][22][23][24][25][26][27]33,44,68,129,130,133,134]. Vinyl group Cys C-S bond (heme-CO-CH 2 -S-CH 2 -C α ) Cyt rb 552 ; S71C cyt b 5 ; N57C/S71C cyt b 5 ;…”