Characterization of Nonsymbiotic Tomato Hemoglobin

Abstract

The nonsymbiotic tomato hemoglobin SOLly GLB1 (Solanum lycopersicon) is shown to form a homodimer of approximately 36 kDa with a high affinity for oxygen. Furthermore, our combined ultraviolet/visible, resonance Raman, and continuous wave electron paramagnetic resonance (EPR) measurements reveal that a mixture of penta- and hexacoordination of the heme iron is found in the deoxy ferrous form, whereas the ferric form shows predominantly a bis-histidine ligation (F8His-Fe(2+/3+)-E7His). This differs from the known forms of vertebrate hemoglobins and myoglobins. We have successfully applied our recently designed pulsed-EPR strategy to study the low-spin ferric form of tomato hemoglobin. These experiments reveal that, in ferric SOLly GLB1, one of the histidine planes is rotated 20 degrees (+/-10 degrees ) away from a N(heme)-Fe-N(heme) axis. Additionally, the observed g-values indicate a quasicoplanarity of the histidine ligands. From the HYSCORE (hyperfine sublevel correlation) measurements, the hyperfine and nuclear quadrupole couplings of the heme and histidine nitrogens are identified and compared with known EPR/ENDOR data of vertebrate Hbs and cytochromes. Finally, the ligand binding kinetics, which also indicate that the ferrous tomato Hb is only partially hexacoordinated, will be discussed in relation with the heme-pocket structure. The similarities and differences with other known nonsymbiotic plant hemoglobins will be highlighted.

Probing the heme-pocket structure of the paramagnetic forms of cytoglobin and a distal histidine mutant using electron paramagnetic resonance

“…The EPR parameters will be compared with the NMR data on ferric wt CYGB [16] and also with our earlier EPR results on the ferric forms of tomato hemoglobin and mouse neuroglobin (mNgb), both proteins showing bis-hisitidine coordination [20,21].…”

“…We recently demonstrated a strategy of combining pulsed and CW EPR techniques to obtain structural and electronic information concerning the heme-pocket environment of ferric low-spin heme proteins, and applied the method successfully to different globins [19][20][21]. In the first part of the present work, we will use this approach to solve the outlined ambiguities about the heme-pocket structure of ferric CYGB.…”

“…In our previous work, we outlined a pulsed EPR strategy that allows determination of the position of the histidine planes in the heme pocket of bis-hisitidine-coordinated lowspin ferric heme proteins [19][20][21]. This strategy involves three steps: (1) of the other systems we studied before [19][20][21].…”

“…This strategy involves three steps: (1) of the other systems we studied before [19][20][21]. Because of this g anisotropy and large g strain effects, the electron-spin-echo intensity at observer positions near g=g x is very low and no X-band HYSCORE spectra could be recorded within realistic measurement times.…”

Non-symbiotic hemoglobin and its relation with hypoxic stress

“…The nsHb of tomato has hexacoordination of the Fe +3 ion and a mixture of pentacoordination and hexacoordination of the Fe +2 ion (Ioanitescu et al, 2005). The AtHb1 of Arabidopsis has a 40% pentacoordination (Bruno et al, 2007;Spyrakis et al, 2011a).…”