Proximal Influences in Two-on-Two Globins:  Effect of the Ala69Ser Replacement on Synechocystis sp. PCC 6803 Hemoglobin

Abstract

The cyanobacterium Synechocystis sp. PCC 6803 (S6803) expresses a two-on-two globin in which His46 (distal side) and His70 (proximal) function as heme iron axial ligands. His46 can be displaced by O 2 , CO, and CN − , among others, whereas His70 is not labile under native conditions. The residue preceding the proximal histidine has been implicated in controlling globin axial ligand reactivity; the details of the mechanism, however, are not well understood, and little information exists for bis-histidyl hexacoordinate proteins. In many vertebrate hemoglobins and in the Synechocystis protein, the position is occupied by an alanine whereas, in myoglobins, it is a serine involved in an intricate hydrogen bond network. We examined the role of Ala69 in S6803 hemoglobin through the effects of an Ala → Ser replacement. The substitution resulted in minor structural perturbations, but the holoprotein's response to temperature-, urea-, and acid-induced denaturation was measurably affected. Enhanced three-state behavior was manifested in the decoupling of heme binding and secondary structure formation. Urea-gradient gel experiments revealed that the stability of the apoprotein was unchanged by the replacement and that a slight alteration of the folding kinetics occurred in the holoproteins. Cyanide-binding experiments were performed to assess trans effects. The apparent rate constant for association decreased two-fold upon Ala69Ser replacement. This deceleration was attributed to a change in the lifetime of a state containing a decoordinated His46. The results demonstrated that, as in vertebrate globins and leghemoglobin, proximal influences operate to determine fundamental dynamic and thermodynamic properties of the protein.Keywords truncated hemoglobin; 2-on-2 globin; hexacoordinate hemoglobin; urea-gradient gel; heme binding; thermodynamic stabilityIron protoporphyrin IX (Fe-PPIX) 1 serves as a cofactor in a large number of essential proteins. In the absence of covalent attachment to the protein matrix via protoporphyrin substituents, the contact between Fe(II)-PPIX (or b heme) and the protein is limited to ligation bond(s) to the iron, hydrogen bonding, van der Waals and electrostatic interactions, and hydrophobic † This study was supported by National Science Foundation grants MCB-091182 and MCB-0349409, National Institutes of Health grant GM-054217, and NASA grant NNG04GN33H (DAV).* To whom correspondence should be addressed. Tel: (814) NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 11. forces. The affinity for the heme group and its chemical properties, such as redox potential, ability to bind various ligands, and propensity for generating reactive oxygen species, are strictly controlled by the protein environment.In many b hemoproteins the iron is endogenously hexacoordinated, with the axial positions of its octahedral geometry filled by two histidine side chains (1). A well-known example is cytochrome b 5 , an electron-transfer protein that does not bind...

The role of the heme distal ligand in the post-translational modification of Synechocystis hemoglobin

“…Although the consequences of the His117Ala replacement clearly go beyond prevention of cross-link formation [14,16,47], a decrease in the bond strength of an endogenous distal ligand upon cross-linking could account for easier ligand switching and the change in spectral appearance.…”

“…S3). For reference, wild-type S6803 rHb-R undergoes a partially reversible unfolding transition with a midpoint temperature (T m ) of 74°C under the same conditions [12,16]. Heme release from the single His46 variants can also be modeled as a two-state process.…”

“…Cyanide binding to wild-type rHb-R results in a sample containing the two heme rotational isomers in a $90:10 ratio (supplementary information Fig. S5A) [16,47] compared to $95:5 in the bis-histidine form [12]. The His46 variants of rHb-R in the cyanomet state exhibited broad 1 H NMR spectra and strongly altered heme isomer ratios of $60:40 in His46Ala and His46Leu ( Fig.…”

“…In ferric (met) S7002 rHb, it affects the rate of backbone amide hydrogen exchange unevenly throughout the structure, with a tendency for acceleration on the distal side of the heme and deceleration on its proximal side [8]. In ferric S6803 rHb, cross-linking results in a moderate increase in the rate of cyanide binding [16]. Heightened resistance to thermal and chemical denaturation is observed in both ferric S7002 and S6803 rHbs [14,16].…”

“…In ferric S6803 rHb, cross-linking results in a moderate increase in the rate of cyanide binding [16]. Heightened resistance to thermal and chemical denaturation is observed in both ferric S7002 and S6803 rHbs [14,16]. Thus, in addition to holding the heme group inside the protein matrix, the histidine-heme covalent attachment alters several essential properties of the protein, and it is of interest to delineate the factors that control its formation.…”