Studying high-spin ferric heme proteins by pulsed EPR spectroscopy: Analysis of the ferric form of the E7Q mutant of human neuroglobin

Abstract

In this work, the high-spin ferric form of the E7Q mutant of human neuroglobin (E7Q-NGB) is studied by X-band continuous-wave electron paramagnetic resonance (CW EPR) and hyperŸ237 sublevel correlation (HYSCORE) spectroscopy. It is shown that the use of matched pulses in the HYSCORE experiment is essential to observe the nitrogen speetral contributions. The validity of approximating the high-spin Fe(IID system (S = 5/2) as an effective S = 1/2 system is tested and the consequences for the HYSCORE simulations are highlighted. Comparative HYSCORE experiments combined with deuterium exchange experiments for aquometmyoglobin and fer¡ E7Q-NGB clearly show that the heme iron of the latter protein is pentacoordinated, tacking the distal water. Furthermore, CW EPR experiments show that, at high pH, the E10K residue is coordinating to the heme iron in this globin. These observations ate corroborated by resonance Raman experiments and could also be reproduced for other E7 mutants of human and mouse neuroglobin. Finally, the proton and nitrogen hyperfine and nuclear quadrupole parameters obtained for ferric E7Q-NGB are discussed in detail.

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

“…It should also be noted that the observed difference in the dihedral angle is close to the margin given by the experimental error of the x-ray data. The extreme flexibility of the heme-pocket region of neuroglobin has been proven using different techniques [44,45,46], including the observation that the principal g values of ferric wt NGB change upon disulfide bridge formation [15] and the fact that the E10Lys can bind to the heme iron in HE7Q NGB at high pH [22]. The heme pocket in wt CYGB is buried deeper in the protein matrix, although the effect of temperature-induced formations of H-bonds to water molecules in the heme region cannot be excluded.…”

“…However, we should stress again that these simulations are based on the Fe-H distances given by the x-ray data. with earlier study on HE7Q NGB [22]. Figure 6f shows the simulated HYSCORE spectra, for which the hyperfine and nuclear-quadrupole parameters from table 2 were used.…”

“…The EPR data of ferric HE7Q CYGB will be compared with our earlier study on the E7Q mutant of human neuroglobin (NGB) [22]. In the last part, we use CW EPR in combination with absorption spectroscopy to determine whether ferrous wt CYGB is capable of capturing the NO generated by the anaerobically grown E. coli cells in which the protein is over-expressed.…”

EPR analysis of cyanide complexes of wild-type human neuroglobin and mutants in comparison to horse heart myoglobin

“…), we observe a clear influence of the point mutations. While ferric wt NGB and F28LNGB are in a low-spin ferric state due to the bis-histidine ligation of the heme iron [8,15,16], ferric H64QNGB is characterized by a high-spin state of the ferric iron with an absence of a distal ligand [37]. However, inspection of Table 1 shows that there is no obvious relationship between the coordination of the ferric haem iron in the absence of exogenous ligands on the one hand and the principal g values after cyanide ligation on the other hand.…”

“…The QuickChangeTM site-directed mutagenesis method (Stratogene) was used to make the F28LNGB and H64QNGB mutants as described earlier [16,37]. Expression of wt NGB and its mutants was done as reported earlier [8].…”