The role of the heme distal ligand in the post-translational modification of Synechocystis hemoglobin.
Synechocystis sp. PCC 6803 hemoglobin is a cyanobacterial Group I truncated hemoglobin. In the absence of an exogenous ligand, its single heme group is coordinated by His46 (E10, distal) and His70 (F8, proximal). The protein can undergo a post-translational modification by which His117 (H16, in the C-terminal helix) reacts with the heme 2-vinyl group to form a Markownikoff adduct. The new C-N bond prevents heme loss, alters the dynamics of the protein, and influences ligand binding to the heme group. To explore the factors conditioning the formation of the cross-link, variants of the protein that contained an alanine or a leucine at position 46 (E10) were prepared. A double replacement (His46Leu and Tyr22 (B10) to Phe) was also performed to perturb the network of interactions stabilizing bound exogenous ligand. The single and double replacements affected the optical and NMR properties of the globin, each in a different fashion. Heme-protein cross-linking, as promoted by sodium dithionite, was retarded by the replacement of His46, but reactivity was recovered when imidazole or cyanide was used as exogenous ligand. In addition, a significant amount of a second product was systematically obtained when dithionite treatment was performed on the cyanide-bound proteins. This species was identified by NMR spectroscopy to be an adduct to the 4-vinyl group. It was concluded that the specificity and rate of the cross-linking reaction depended critically on the nature of the sixth ligand to the heme iron.
Nothnagel
Henry J
HJ
TC Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.
Love
Nicole
N
Lecomte
Juliette T J
JT
D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D011499:Protein Processing, Post-Translational; D046939:Synechocystis / Q000378:metabolism*; D054793:Truncated Hemoglobins / Q000737:chemistry* / Q000378:metabolism*
D008024:Ligands; D054793:Truncated Hemoglobins; D006418:Heme