Spectroscopic analysis of moss (Ceratodon purpureus and Physcomitrella patens) recombinant non-symbiotic hemoglobins

Abstract

Non-symbiotic hemoglobins (nsHbs) are O₂-binding proteins widely distributed in land plants, including primitive bryophytes. Little is known about the properties of bryophyte nsHbs. Here, we report the spectroscopic characterization of two moss recombinant nsHbs, CerpurnsHb of Ceratodon purpureus and PhypatnsHb of Physcomitrella patens. Spectra showed that the absorption maxima of the ferrous and ferric forms of recombinant CerpurnsHb are located at 418, 531 and 557 nm and 407, 537, 569 (shoulder) and 632 (shoulder) nm, respectively, and of PhypatnsHb are located at 422, 529 and 557 nm and 407, 531, 571 (shoulder) and 647 (shoulder) nm, respectively. These absorption maxima are similar to those of rice Hb1. Also, the absorption maxima of the oxygenated ferrous form of recombinant CerpurnsHb and PhypatnsHb are located at 412, 541 and 575 nm and 414, 541 and 574 nm, respectively, similar to those of oxygenated rice Hb1 and cowpea leghemoglobin II. This evidence indicates that CerpurnsHb and PhypatnsHb are mostly hexacoordinate and that they bind O₂.

Keywords: :

• Fe-coordination
• hemoglobin
• moss
• non-symbiotic
• recombinant proteins
• visible spectroscopy

Non-symbiotic hemoglobins (nsHbs) are O₂-binding proteins widely distributed in land plants, from primitive bryophytes to evolved monocots and dicots.¹ Based on O₂-affinity and sequence similarity nsHbs are classified into type 1 and type 2 (nsHbs-1 and nsHbs-2, respectively).^2,3 The O₂-affinity of nsHbs-1 is very high because of an extremely low O₂-dissociation rate constant.^3-5 Analyses by X-ray crystallography, site-directed mutagenesis and visible spectroscopy revealed that the extremely low O₂-dissociation rate constant of nsHbs-1 primarily results from Fe-heme hexacoordination by distal His.^4,6 Absorption spectra of nsHbs-1 are similar to those of other Hbs, however nsHbs-1 exhibit distinctive absorption maxima in the deoxyferrous (Hb²⁺) form. Specifically, nsHb-1²⁺ exhibits peaks at ~526 and ~556 nm, which is characteristic of hexacoordinate Hbs.^4,7 In contrast, pentacoordinate Hbs exhibit a broad peak centered at ~556 nm in their Hb²⁺ form.^7,8

Little is known about the biophysical properties of primeval land plant nsHbs (e.g., the postulated ancestors of nsHbs-1 and nsHbs-2 ¹), such as bryophyte nsHbs. To better understand these nsHbs, the structure of a moss (Ceratodon purpureus) nsHb (CerpurnsHb) was modeled.⁹ The predicted structure suggested that Fe-heme in this protein is hexacoordinate, however this observation has not been verified experimentally. Here, we report the generation and spectroscopic characterizations of recombinant CerpurnsHb and Physcomitrella patens nsHb (PhypatnsHb).

We generated recombinant CerpurnsHb and PhypatnsHb from plasmids pCR2.1::CerHb⁹ and pCRII::B46,¹⁰ essentially as described by Arredondo-Peter et al.^4,8 Inserts were subcloned into the plasmid pET28b (Novagen), generating constructs pET28b::CerpurnsHb and pET28b::PhypatnsHb, then transformed into E. coli Tuner(DE3)pLacI. The inserts within plasmids pET28b::CerpurnsHb and pET28b::PhypatnsHb were fully sequenced. Soluble extracts were obtained from recombinant E. coli pET28b::CerpurnsHb and pET28b::PhypatnsHb, and recombinant CerpurnsHb and PhypatnsHb were detected by SDS-PAGE. Soluble extracts were subjected to spectroscopic analysis using soluble extracts from untransformed E. coli Tuner(DE3)pLacI as blanks. Ferrous Hb was oxidized to ferric (Hb³⁺) Hb by adding potassium ferricyanide; Hb²⁺ was formed by adding sodium dithionite; and air was bubbled through the Hb²⁺ solution to generate the O₂-ligated (Hb²⁺O₂) form of Hb.

DNA sequencing detected no mutations within the inserts of plasmids pET28b::CerpurnsHb and pET28b::PhypatnsHb. Thus, the sequences of recombinant CerpurnsHb and PhypatnsHb were identical to that predicted by the pCR2.1::CerHb and pCRII::B46 plasmids. SDS-PAGE analysis showed that recombinant CerpurnsHb and PhypatnsHb of the expected molecular masses (19.6 and 19.9 KD, respectively) were synthesized by E. coli Tuner(DE3)pLacI (Fig. 1). Spectra of recombinant CerpurnsHb and PhypatnsHb were highly similar to those of other Hbs (Fig. 2). The absorption maxima of Hb²⁺ and Hb³⁺ forms of recombinant CerpurnsHb are located at 418, 531 and 557 nm and 407, 537, 569 (shoulder) and 632 (shoulder) nm, respectively, and in PhypatnsHb at 422, 529 and 557 nm and 407, 531, 571 (shoulder) and 647 (shoulder) nm, respectively, similar to those of rice Hb1. Also, the absorption maxima of the Hb²⁺O₂ form of recombinant CerpurnsHb and PhypatnsHb were located at 412, 541 and 575 nm and 414, 541 and 574 nm, respectively, similar to those of oxygenated rice Hb1 and cowpea LbII (Table 1). This evidence indicates that CerpurnsHb and PhypatnsHb are hexacoordinate and that they bind O₂. However, the 531 nm maximum of CerpurnsHb²⁺ and 529 nm maximum of PhypatnsHb²⁺ are weak compared with the maximum of rice Hb1.⁴ This observation suggests that hexacoordination is partial in both CerpuprnsHb²⁺ and PyspatnsHb²⁺ and that these proteins may exist in a mixture of hexa- and pentacoordinate forms. Thus, it is likely that the O₂-affinities of CerpurnsHb and PhypatnsHb are higher than those reported for other hexacoordinate land plant nsHbs. An unusual characteristic of the oxygenated CerpurnsHb²⁺ and PhypatnsHb²⁺ spectra was the existence of absorption peaks at 645 nm (Fig. 2 and Table 1). These spectra are similar to that from recombinant human histoglobin obtained from E. coli grown in a fermentation apparatus aerated using pure oxygen.¹¹ This observation suggests that Fe-heme is in the high-spin form in oxygenated CerpurnsHb²⁺ and PhypatnsHb²⁺, however the origin of peaks at 645 nm is not known. The results reported here provide knowledge of the spectroscopic properties of bryophyte nsHbs and corroborate the Fe-heme hexacoordination predicted for modeled CerpurnsHb.⁹

Figure 1. Detection of recombinant CerpurnsHb and PhypatnsHb synthesized by E. coli Tuner(DE3)pLacI. Aliquots (~30 μg of total protein) were separated by SDS-PAGE in a 15% gel. Line 1, molecular mass markers; lines 2 and 3, soluble extract of the untransformed E. coli Tuner(DE3)pLacI; line 4, soluble extract of the E. coli Tuner(DE3)pLacI transformed with the pET28b::CerpurnsHb construct; line 5, soluble extract of the E. coli Tuner(DE3)pLacI transformed with the pET28b::PhypatnsHb construct. Arrows indicate the recombinant CerpurnsHb and PhypatnsHb. Markers are shown in kD.

Figure 2. Absorption spectra of E. coli Tuner(DE3)pLacI soluble extracts containing the recombinant CerpurnsHb (A) and PhypatnsHb (B). Blue lines, Hb³⁺ form; red lines, Hb²⁺O₂ form; and black lines, Hb²⁺ form.

Table 1. Spectral characteristics of C. purpureus and P. patens recombinant nsHbs and hexacoordinate rice Hb1⁴ and human neuroglobin,¹² and pentacoordinate cowpea LbII⁸

	Absorption maxima (nm)
State/ligand	Søret region	Q region
CerpurnsHb
Ferrous deoxygenated	418	531		557
Ferrous oxygenated	412		541		575	645
Ferric	407		537		569 (shoulder)	632 (shoulder)
PhypatnsHb
Ferrous deoxygenated	422	529		557
Ferrous oxygenated	414		541		574	645
Ferric	407		531		571 (shoulder)	647 (shoulder)
Rice Hb1
Ferrous deoxygenated	424	529		557
Ferrous oxygenated	412		540		576
Ferric	410		540	556 (shoulder)
Human neuroglobin
Ferrous deoxygenated	425	527		563
Ferrous oxygenated	413		542		579
Ferric	417		538		567 (shoulder)
Cowpea Lbll
Ferrous deoxygenated	428			556
Ferrous oxygenated	411		540		574
Ferric	404		534	560 (shoulder)		620 (shoulder)

Abbreviations:
CerpurnsHb	=	Ceratodon purpureus non-symbiotic hemoglobin
Hb	=	hemoglobin
Lb	=	leghemoglobin
nsHb	=	non-symbiotic hemoglobin
PhypatnsHb	=	Physcomitrella patens non-symbiotic hemoglobin

Acknowledgments

Authors wish to express their gratitude to Miss Gillian Klucas for English corrections. Work in the authors´ laboratory has been funded by SEP-PROMEP (grant no. UAEMor-PTC-01–01/PTC23) and Consejo Nacional de Ciencia y Tecnología (CoNaCyT grant nos. 25229N and 42873Q), México. C. V.-L. is a postdoctoral fellow supported by CoNaCyT.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.