Comparison of the Crystal and Solution Structures of a Cyanobacterial Hemoglobin

Abstract

GlbN is a monomeric 2/2 hemoglobin found in several cyanobacteria. Physiological data suggest that, in Synechococcus sp. PCC 7002, GlbN protects the cell from damage associated with nitrate metabolism [1]. Under reducing conditions, GlbN undergoes a facile posttranslational cross-linking between His117 and the heme 2-vinyl. In a study of the enzymatic and chemical properties of GlbN, the solution structure of the protein in the ferric, bis-histidine state with heme posttranslational modification was determined by NMR methods. As this protein is paramagnetic (S = 1/2) and can be prepared in the reduced state (S = 0), heme methyl hyperfine chemical shifts were used to determine the orientation of the ligating histidines, and proton pseudocontact shifts were applied in the refinement. The NMR model contains regions that appear to sample multiple conformations, specifically the A helix and EF loop. Here we present the X-ray structure of GlbN. Crystals were obtained by the vapor diffusion hanging drop method, and data were collected under cryogenic conditions (National Synchrotron Light Source, BNL). The structure of the related Synechocystis sp. PCC 6803 GlbN (1RTX) was used as the search model for molecular replacement. Iterative model building yielded a final structure at 1.65 A resolution. The X-ray data revealed a ruffled heme, provided a reliable representation of its environment in solution, and allowed for an assessment of the paramagnetic NMR approach. The X-ray data also gave an indication of the effect of crystal packing on the flexible regions of GlbN. Comparison with the Synechocystis GlbN structure highlighted the resilience of the 2/2 globin fold as well as subtle differences in the solvent accessibility of the heme active site.[1] Scott et al. (2010) Biochemistry 49:7000Supported by NSF MCB 0843439