Lysine as a heme iron ligand: A property common to three truncated hemoglobins from Chlamydomonas reinhardtii

Abstract

Background: The nuclear genome of Chlamydomonas reinhardtii encodes a dozen hemoglobins of the truncated lineage. Four of these, named THB1-4, contain a single ~130-residue globin unit. THB1, which is cytoplasmic and capable of nitric oxide dioxygenation activity, uses a histidine and a lysine as axial ligands to the heme iron. In the present report, we compared THB2, THB3, and THB4 to THB1 to gain structural and functional insights into algal globins.

Methods: We inspected properties of the globin domains prepared by recombinant means through site-directed mutagenesis, electronic absorption, CD, and NMR spectroscopies, and X-ray crystallography.

Results: Recombinant THB3, which lacks the proximal histidine but has a distal histidine, binds heme weakly. NMR data demonstrate that the recombinant domains of THB2 and THB4 coordinate the ferrous heme iron with the proximal histidine and a lysine from the distal helix. An X-ray structure of ferric THB4 confirms lysine coordination. THB1, THB2, and THB4 have reduction potentials between -65 and -100 mV, are capable of nitric oxide dioxygenation, are reduced at different rates by the diaphorase domain of C. reinhardtii nitrate reductase, and show different response to peroxide treatment.

Conclusions: Three single-domain C. reinhardtii hemoglobins use lysine as a distal heme ligand in both Fe(III) and Fe(II) oxidation states. This common feature is likely related to enzymatic activity in the management of reactive oxygen species.

General significance: Primary structure analysis of hemoglobins has limited power in the prediction of heme ligation. Experimental determination reveals variations in this essential property across the superfamily.

Keywords: 2-over-2 hemoglobin; Heme reduction potential; Nitric oxide dioxygenase; Nuclear magnetic resonance.

Fig. 1.

(a) The X-ray structure of ferric THB1 (PDB ID: 4XDI, [5]) showing the proximal histidine (H77 or F8 in Perutz helical notation), distal lysine (K53 or E10), and tyrosine 29 (B10). THB1 has the typical truncated or 2-over-2 hemoglobin fold [8]. Helices are labeled A to C and E to H. (b) The heme group and nomenclature used in the text. The porphyrin ring supplies four pyrrole nitrogen ligands to the iron. Within a protein, the heme group can be found with no axial ligand (four-coordinate), one axial ligand (five- or pentacoordinate), or two axial ligands (six- or hexacoordinate). Typical iron oxidation states for a hemoglobin are Fe(II) (ferrous) and Fe(III) (ferric).

Fig. 2.

The primary structure of C. reinhardtii THB1–4 and C. eugametos LI637 (CtrHb). In Perutz notation, key positions (bold) are tyrosine “B10” (Y29, Y46, Y167, Y52, and Y63, respectively), the “E10” residue (distal, K53, K70, H191, K76, and K87, respectively), and the “F8” residue (proximal, H77, H94, Q215, H100, and H111, respectively). Four cysteines are underlined in THB3. Italics are used for the portions of sequence that were not included in the expressed recombinant proteins. The letters above the top sequence refer to the helices present in the X-ray structure of THB1 (Fig. 1a).

Fig. 3.

Absorption spectra of 10–15 μM THB2 (a), THB3-S4 (b), and THB4 (c) in 100 mM (a, c) or 20 mM (b) Tris, pH 7.5. Fe(III) spectra are shown with solid lines and Fe(II) spectra are shown with dashed lines. The intensity of the region from 480–650 nm was magnified by a factor of 5. Extinction coefficients for THB2 and THB4 were obtained with the hemochromogen assay. Because of weak heme binding to THB3-S4 and unknown stoichiometry of the reconstituted material, the y-axis in c is absorbance.

Fig. 4.

NMR spectra of THB2 in the Fe(II) state at 25 °C. The heme orientational isomers are denoted A and B. (a) 1D ¹H spectrum of THB2 at pH 10.0 in 10% ²H₂O / 90% ¹H₂O with resolved K70 resonances labeled. (b) Upfield region of ¹H-¹H NOESY data showing K70 resonances. (c) Portion of ¹H-¹H TOCSY data showing some of the connectivities of K70. (d) Portion of ¹H-¹H NOESY spectrum of Y46F THB2 at pH 10.0 in 100% ²H₂O showing dipolar contacts between the heme meso protons (top labels) and K70. One of the K70 ε protons in the A form has contacts with the δ-meso proton of the heme, while the corresponding K70 ε proton in the B form contacts the β-meso proton of the heme, consistent with heme orientational isomerism. Assignment to specific protons of K70 is tentative, except for NζHs.

Fig. 5.

NMR spectra of THB4 in the ferrous state in 10% ²H₂O / 90% ¹H₂O at 25 °C. (a) 1D ¹H spectrum of THB4 at pH 10.0, showing the upfield resonances characteristic of lysine coordination, between −1.1 and −8.2 ppm. The NζH₂ of K76 is indicated at −8.1 ppm. Inset: Region of the ¹H-¹⁵N HSQC spectrum at pH 7.5 showing the K76 NζH₂ resonances. (b) Upfield portion of the ¹H-¹H NOESY of THB4 at pH 10.0, showing some of the K76 dipolar contacts to itself and to Phe65.

Fig. 6.

(a) Ribbon diagram of THB4 (PDB ID; 6BME, chain A) with helices labeled. The axial heme ligands and Tyr B10 are indicated. (b,c): 2 F_o − F_c electron-density map (contoured at the 1.0 σ level, chain A) of the heme and axial ligands. The electron density maps were prepared with PyMOL [49].

Fig. 7.

Comparison of the heme binding pockets of (a) THB4 (PDB ID: 6BME), (b) THB1 (PDB ID: 4XDI), and (c) Synechococcus GlbN (PDB ID: 4MAX) showing the axial ligands (E10 and F8), key residues B10, E7, and E11, and water molecules near residue B10 or E10.

Fig. 8.

Reduction potential measurements using I3S as the dye at pH 7.0 and 25 °C. (a) THB1; (b) THB2; (c) THB4. Values are obtained by adding the y-intercept of the linear fit to −81 mV. Inspection shows the ranking E_m(THB4) < E_m(THB2) < E_m(THB1).

Fig. 9.

Reduction of THB1 (solid line), THB2 (long dashed line) and THB4 (short dashed line) by the heme-less recombinant diaphorase domain of NIT1 (pH 7.5 and 25 °C). The fraction of reduced protein was determined by following change in absorbance value of the Soret maximum. Conditions were identical for the three proteins.

Fig. 10.

Reaction of (a) THB1, (b) THB2 and (c) THB4 with NO (pH 7.5 and 25 °C). The reaction was followed by observing the absorbance of the sample at 545 nm, which reports on the proportions of Fe(II)-O₂ and Fe(III) species. In each panel, the bottom dashed line indicates the absorbance of the starting Fe(III) state; the top dashed line indicates the absorbance of the Fe(II)-O₂ state; and vertical arrows indicate NO additions.