Distinctive structural properties of THB11, a pentacoordinate Chlamydomonas reinhardtii truncated hemoglobin with N- and C-terminal extensions

Abstract

Hemoglobins (Hbs) utilize heme b as a cofactor and are found in all kingdoms of life. The current knowledge reveals an enormous variability of Hb primary sequences, resulting in topological, biochemical and physiological individuality. As Hbs appear to modulate their reactivities through specific combinations of structural features, predicting the characteristics of a given Hb is still hardly possible. The unicellular green alga Chlamydomonas reinhardtii contains 12 genes encoding diverse Hbs of the truncated lineage, several of which possess extended N- or C-termini of unknown function. Studies on some of the Chlamydomonas Hbs revealed yet unpredictable structural and biochemical variations, which, along with a different expression of their genes, suggest diverse physiological roles. Chlamydomonas thus represents a promising system to analyze the diversification of Hb structure, biochemistry and physiology. Here, we report the crystal structure, resolved to 1.75 Å, of the heme-binding domain of cyanomet THB11 (Cre16.g662750), one of the pentacoordinate algal Hbs, which offer a free Fe-coordination site in the reduced state. The overall fold of THB11 is conserved, but individual features such as a kink in helix E, a tilted heme plane and a clustering of methionine residues at a putative tunnel exit appear to be unique. Both N- and C-termini promote the formation of oligomer mixtures, and the absence of the C terminus results in reduced nitrite reduction rates. This work widens the structural and biochemical knowledge on the 2/2Hb family and suggests that the N- and C-terminal extensions of the Chlamydomonas 2/2Hbs modulate their reactivity by intermolecular interactions.

Keywords: Ligand tunnels; Nitrite reduction; Oligomerization; Pentacoordination; Truncated hemoglobin.

Fig. 1

The crystal structure of the THB11 HBD reveals the typical class I 2/2Hb structure, but a kink in helix E and a tilt of the heme group. a Cartoon diagram of THB11-NC (PDB: 6TD7). Helices are colored individually and labeled according to the Perutz nomenclature [5]. b The structures of the indicated cyanomet 2/2Hbs (THB1: C. reinhardtii THB1, PDB: 6CII; Syn GlbN: Synechocystis 2/2Hb, PDB: 1S69 chain A; LI637: C. eugametos 2/2Hb LI637, PDB: 1DLY chain A; Mt GlbN: M. tuberculosis 2/2Hb, PDB: 1RTE chain B) were aligned to THB11-NC. The view is from the side of the A helix and the EF-turn, respectively. The heme groups, coordinating HisF8 residues and CN^– ligands are shown for all proteins. For orientation, the tertiary structure of THB11-NC is depicted in transparent gray. The figures were generated in PyMOL

Fig. 2

The heme environment of THB11-NC. a The top view shows the moderately staggered and tilted imidazole ring of the heme-coordinating HisF8(81) residue and the H-bond distance between the HisF8(81) Nδ-atom and the backbone carbonyl group of ValF4(77). b The heme propionates are in polar contact distance to ArgF10(83), Lys75 (EF-loop) and water molecules. The highly conserved heme-shielding PheE14(60) is also shown. c The side view shows heme-Fe ligand distances as well as distal site residues reported to play a role in ligand stabilization by H-bond networks in class I 2/2Hbs (B10, E7, E11, represented by Tyr32, Leu53 and Gln57 in THB11-NC) as well as LysE10(56). a–c Figures were created and distances measured in PyMOL. Putative polar contact distances are indicated by dashed lines

Fig. 3

Possible ligand access routes can be identified in THB11-NC. a Tunnels that start at the heme-Fe of Chlamydomonas (Cr) THB11-NC (PDB: 6TD7) and M. tuberculosis (Mt) GlbN (PDB: 1RTE, chain B) were computed by Caver 3.0.1, employing a minimal probe radius of 0.9, and visualized in PyMOL. Residues reported to line the long (LT) and short tunnels (ST) are represented as sticks and, in panel b, are labeled according to the Perutz nomenclature, including their position in the respective sequences. HA: heme access site observed in THB11-NC

Fig. 4

THB11 length variants form different oligomeric states. a Anion exchange chromatography (AEC) profiles of the indicated protein variants, which had been purified from E. coli cell lysates by Strep-tag affinity chromatography before. The brackets above the peaks indicate the elution fractions that were combined for subsequent analyses [black, blue: peak (P) 1, 2]. The molecular mass of each protein calculated from the primary sequence are indicated to the left. b Denaturing (SDS) and native gel electrophoreses of the protein solutions collected from the AEC peaks P1 and P2. The numbered bands observed in the native gels were cut out and subsequently subjected to denaturing gel electrophoresis. c Size exclusion chromatography (SEC) profiles of the proteins present in the two AEC peaks. kDa labels within the SEC graphs indicate the molecular masses calculated from the elution volumes according to the column’s calibration curve, colors indicate the original AEC P1 (black) or P2 (blue). d UV–Vis spectra of the protein solutions collected from the two AEC peaks, P1 and P2. The spectra were normalized with regard to the Soret maxima (set to 1)

Fig. 5

Nitrite reduction rates of THB11 length variants. Recombinant deoxy Chlamydomonas THB11 length variants as well as horse heart Mb and Chlamydomonas THB1 at concentrations of 5 µM heme were incubated in anoxic 50 mM HEPES buffer, pH 7.4, 100 mM NaCl, supplemented with 600 µM NaDt and 0.25 to 1 mM potassium nitrite. The decrease of the Soret peak maxima of deoxy (heme-Fe^II) globins was followed spectroscopically at 20 °C. Pseudo first order rate constants k_obs were obtained from exponential decay fits of the absorption changes in time (see Fig. S5). Linear fits of the k_obs mean values obtained at different nitrite concentrations yielded the bimolecular reaction rates. Experiments were done in technical triplicates per nitrite concentration from at least two independent protein batches. The bars indicate the averages of reaction rates calculated per experiment, and error bars indicate the standard deviation. FL full length, -N without N terminus, -C without C terminus, -NC without N- and C-termini