Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium synechocystis sp. PCC 6803

Abstract

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a gene~slr2097, glbN ! encoding a 123 amino-acid product with sequence similarity to globins. Related proteins from cyanobacteria, ciliates, and green algae bind oxygen and have a pronounced tendency to coordinate the heme iron with two protein ligands. To study the structural and functional properties of Synechocystis sp. PCC 6803 hemoglobin, slr2097 was cloned and overexpressed in Escherichia coli. Purification of the hemoglobin was performed after addition of hemin to the clarified cell lysate. Recombinant, heme-reconstituted ferric Synechocystis sp. PCC 6803 hemoglobin was found to be a stable helical protein, soluble to concentrations higher than 500 mM. At neutral pH, it yielded an electronic absorption spectrum typical of a low-spin ferric species, with maxima at 410 and 546 nm. The proton NMR spectrum revealed sharp lines spread over a chemical shift window narrower than 40 ppm, in support of low-spin hexacoordination of the heme iron. Nuclear Overhauser effects demonstrated that the heme is inserted in the protein matrix to produce one major equilibrium form. Addition of dithionite resulted in an absorption spectrum with maxima at 426, 528, and 560 nm. This reduced form appeared capable of carbon monoxide binding. Optical data also suggested that cyanide ions could bind to the heme in the ferric state. The spectral properties of the putative Synechocystis sp. PCC 6803 hemoglobin confirmed that it can be used for further studies of an ancient hemoprotein structure.

Covalent heme attachment in Synechocystis hemoglobin is required to prevent ferrous heme dissociation

“…This is the case for ferric (but not ferrous) Mb, in which transfer to the heme-scavenger protein (apoH64Y/V68F Mb) has been used to measure rate constants for heme dissociation (Hargrove et al 1996b), demonstrating that this protein loses heme at a rate of ;0.007 h À1 at pH 7.0. Previous work demonstrated very slow heme loss from ferric wild-type SynHb without the covalent bond (Scott and Lecomte 2000;Lecomte et al 2001). Figure 6 shows heme dissociation experiments for SynHb (containing the covalent bond) and the SynH117A mutant protein in both the ferrous and ferric oxidation states.…”

“…Another nontraditional group of Hbs, the ''truncated'' Hbs (trHbs), is found in eubacteria, bacteria, single-celled eukaryotes, and plants Scott and Lecomte 2000;Hvitved et al 2001;Watts et al 2001;Wittenberg et al 2002). They can be 20-40 residues shorter than the majority of Hbs and comprise an abbreviated globin architecture surrounding the heme that forms a ''2-on-2'' fold (Pesce et al 2000;Wittenberg et al 2002).…”

“…Prior to identification of this bond, the existence of two forms of the protein had been noted, and experiments were carefully performed only on the form of the protein that, in retrospect, lacked the covalent bond (Lecomte et al 2001). These experiments included observation of slow heme release (Scott and Lecomte 2000;Lecomte et al 2001), thermal denaturation (Lecomte et al 2001), and measurement of the electrochemical midpoint potential (Lecomte et al 2001). After the covalent bond was discovered as the source of heterogeneity, partial NMR structural analysis indicated little global conformational change among the ferric tertiary structures of the two forms of wild-type protein and the H117A mutant protein (which is incapable of forming the covalent bond) (Falzone et al 2002;Vu et al 2004aVu et al , 2004b.…”

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

“…PCC 6803 (S6803) and Synechococcus sp. PCC 7002 (S7002), exhibit bis-histidyl coordination of the heme iron in the absence of exogenous ligand [9][10][11]. In these two proteins, His70 (F8) assumes the traditional role of kinetically stable axial ligand on the proximal side.…”

Structure and Reactivity of Hexacoordinate Hemoglobins

“…HxHbs have been found in plants, animals, and cyanobacteria [18][19][20]. They were first noted in the plant "nonsymbiotic" hemoglobins (nsHbs), which were discovered during a search for globins in plants that are unrelated to oxygen transport [18].…”