Structural investigations of the hemoglobin of the cyanobacterium Synechocystis PCC6803 reveal a unique distal heme pocket

Abstract

A putative hemoglobin (Hb) gene, related to those previously characterized in the green alga Chlamydomonas eugametos, the ciliated protozoan Paramecium caudatum, the cyanobacterium Nostoc commune and the bacterium Mycobacterium tuberculosis, was recently discovered in the complete genome sequence of the cyanobacterium Synechocystis PCC 6803. In this paper, we report the purification of Synechocystis Hb and describe some of its salient biochemical and spectroscopic properties. We show that the recombinant protein contains Fe-protoporphyrin IX and forms a very stable complex with oxygen. The oxygen dissociation rate measured, 0.011 s 21 , is among the smallest known and is four orders of magnitude smaller than the rate measured for N. commune Hb, which suggests functional differences between these Hbs. Optical and resonance Raman spectroscopic study of the structure of the heme pocket of Synechocystis Hb reveals that the heme is 6-coordinate and low-spin in both ferric and ferrous forms in the pH range 5.5±10.5. We present evidence that His46, predicted to occupy the helical position E10 based on amino-acid sequence comparison, is involved in the formation of the ferric and ferrous 6-coordinate low-spin structures. The analysis of the His46Ala mutant shows that the ferrous form is 5-coordinate and high-spin and the ferric form contains a 6-coordinate high-spin component in which the sixth ligand is most probably a water molecule. We conclude that the heme pocket of the wild type Synechocystis Hb has a unique structure that requires a histidine residue at the E10 position for the formation of its native structure. [3,6]. Despite this considerable divergence at the amino-acid sequence level, the trHbs are predicted to display a globin fold albeit with substantial residue deletions at either N-or C-terminus and in the CD±D region.Sequence comparisons show that the trHbs possess several of the key residues that are required for ligand binding in vertebrate and nonvertebrate Hbs. These include the proximal histidine at position F8 and the distal residue at position E7, which is almost invariably a histidine or glutamine. In vertebrate and nonvertebrate Hbs, the proximal histidine anchors the heme to the polypeptide chain while the distal E7 residue stabilizes oxygen through hydrogen bonding [7,8]. In a single instance, a residue (Leu) not capable of hydrogen bonding was found at the E7 position in M. tuberculosis trHb [5]. Another distal residue, at position B10, which is invariably tyrosine in trHbs, except for that of N. commune with a histidine residue, has been shown to affect ligand stability by further hydrogen bonding in the trHbs from C. eugametos [9] and M. tuberculosis [5]. A similar role has been shown for the B10 tyrosine residue in the Hb of the nematode Ascaris suum [10,11]. It is notable that C. eugametos and P. caudatum trHbs with the same B10 and E7 distal residues have very different O 2 affinities (P 50 , 0.005 mmHg and 0.6 mmHg, for C. eugametos [9] and P. caudatum [12] trHbs, respectively). This s...

Protein structure in the truncated (2/2) hemoglobin family

“…As mentioned above, Ss-2/2HbN displays bis-histidine heme hexacoordination, where HisF8 and HisE10 are the Fe-atom ligands (21,22,32). Binding of an exogenous ligand to the heme distal site, requires the dissociation of HisE10 from the heme and remarkable conformational changes affecting the B-and E-helices (12,33).…”

“…The hexacoordinate Synechocystis sp. 2/2HbN displays a covalent bond linking HisH16 and the 2-vinyl group of the heme, that may modulate the reactivity of the heme group (10,11,21,22). Moreover, heme isomerism has been reported in some of the 2/2Hb crystal structures (23).…”

Purification and Spectroscopic Characterization of Ctb, a Group III Truncated Hemoglobin Implicated in Oxygen Metabolism in the Food-Borne Pathogen Campylobacter jejuni

“…This trait is displayed in a number of Hbs including trHbs found in M. tuberculosis (26,35), Chlamydomonas (25), Paramecium (37) and the flavoHb Hmp of E. coli (31). Only the trHb found in Synechocystis is six-coordinate and low-spin when reduced (39).…”

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

“…Compared with the wild-type protein, Met66 CεH 3 was shifted upfield by ∼ 0.2 ppm, a displacement that is also observed in H117A rHb-R and, therefore, cannot be attributed to H-bonding of Ser69 to His70 or the 7-propionate with certainty. NOEs between the heme 8-CH 3 and Tyr65, heme 2-vinyl and Phe84, heme 2-vinyl and Tyr53, heme 4-vinyl and Phe34, heme 3-CH 3 and Leu79, and heme 3-CH 3 and Val87 all confirmed similar placement of the heme. The relative proportions of native protein populating two states that differ by a 180°r otation about the heme α-γ meso axis is 95:5 in the wild-type hemichrome (4).…”

“…The proximal histidine (His70) NδH was found to be in dipolar contact with Met66 CαH, as observed for the wild-type and H117A hemichromes. Met66 assumed the same position with respect to the heme group and the flanking Tyr65, as evidenced by contacts involving Met66 CεH 3 , Tyr65 CεHs, and the heme 8-CH 3 . Compared with the wild-type protein, Met66 CεH 3 was shifted upfield by ∼ 0.2 ppm, a displacement that is also observed in H117A rHb-R and, therefore, cannot be attributed to H-bonding of Ser69 to His70 or the 7-propionate with certainty.…”