Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity
Abstract
:1. Introduction
2. Results
2.1. Equilibrium Conformations of Arabidopsis Hemoglobins (AHbs) in the Absence of Distal His
2.2. Nitrite Reductase Activity of AHbs Mutants
2.3. pH-Dependent Nitrite Reductase Activity of AHb Mutants
2.4. Detection of NO Gas by Chemiluminescence
2.5. Reaction of Nitrosyl AHbs with Peroxynitrite and H2O2
2.6. Sequence Analysis of AHbs and Homology Model of AHb2
3. Discussion
4. Materials and Methods
4.1. Reagents and AHb Preparations
4.2. Reaction of AHbs with Nitrite
4.3. NO Gas Detection by Chemiluminescence
4.4. Release of Nitric Oxide from Nitrosyl-AHb by Oxidizing Agents
4.5. Statistical Analysis
4.6. Sequence Alignment and Homology Modeling
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
Hb | Hemoglobin |
AHb | Arabidopsis thaliana hemoglobin |
Ngb | Neuroglobin |
NO | Nitric oxide |
NO2− | Nitrite |
ROS | Reactive oxygen species |
kobs | Observed rate constant |
kd | Dissociation rate constant |
E7 | Seventh amino acid at E-helix in Hb |
Fe2+ | Ferrous |
Fe3+ | Ferric |
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AHb Proteins | Ligation State | pH | pK | Soret | Visible Bands |
---|---|---|---|---|---|
nm | nm | ||||
AHb1 H69L | |||||
Ferric | Five-coordinate | 6–9 | 390 | 504/637 | |
Ferrous-deoxy | Five-coordinate | 6–9 | 435 | 540/569 | |
AHb2 H66L | |||||
Ferric | Five/six-coordinate | 6 | 7.2 | 404 | 500/631 |
8 | 408 | 532/562 | |||
11 | 10 | 409 | 535/568 | ||
Ferrous-deoxy | Five/six-coordinate | 6 | 422/436 | 560 | |
11 | 10 | 422 | 527/557 | ||
AHb2 H66L-K69L | |||||
Ferric | Five-coordinate | 6–11 | 397 | 498/624 | |
Ferrous-deoxy | Five-coordinate | 6–11 | 420/436 | 538/560 |
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Kumar, N.; Astegno, A.; Chen, J.; Giorgetti, A.; Dominici, P. Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity. Int. J. Mol. Sci. 2016, 17, 640. https://doi.org/10.3390/ijms17050640
Kumar N, Astegno A, Chen J, Giorgetti A, Dominici P. Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity. International Journal of Molecular Sciences. 2016; 17(5):640. https://doi.org/10.3390/ijms17050640
Chicago/Turabian StyleKumar, Nitin, Alessandra Astegno, Jian Chen, Alejandro Giorgetti, and Paola Dominici. 2016. "Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity" International Journal of Molecular Sciences 17, no. 5: 640. https://doi.org/10.3390/ijms17050640