Characterization and Application of Heme Protein-Based Nitric Oxide Donors from Bloodsucking Insects
Zusammenfassung der Projektergebnisse
Nitric oxide (NO) is a key molecule for the regulation of many biological processes in the human body. In the cardiovascular system, NO promotes the relaxation of smooth muscle cells, thus increasing blood vessel diameter and decreasing blood pressure. Other targets are, for example, platelets, thus preventing blood coagulation. Because of the effect of NO to increase blood vessel diameter and to inhibit blood coagulation, it was introduced in the late 19th century as a drug which was applied in the form of nitroglycerin. However, the identification of NO being the actual active compound and the elucidation of its central role in cardiovascular function in general happened much later and was awarded with the Nobel Prize in 1998. Many attempts have been made since then to introduce more synthetic NO donors with new potentials and specificities but no compound has been introduced into the clinic. The major problems are usually the fast decomposition in a biological environment and the lack of sufficient cell or tissue specificity. Nitrophorins (NPs) comprise a unique class of ferriheme proteins originating from the saliva of the blood feeding insect Rhodnius prolixus. The major biological function of these proteins is the transport and delivery of NO from the insect saliva to the blood vessels of a host species where it acts as a vasodilator and blood-coagulation inhibitor. The NO transport is accomplished through the binding of NO to a heme iron. The protein undergoes a significant conformational change when moved from the acidic saliva (pH 5 to 6) to those of the blood plasma (pH ~7.4) which decreases the affinity for NO. It is our aim to understand the underlying molecular mechanisms in detail to learn how NO can successfully applied to living organisms. We demonstrated that ferriheme NPs are not only able to transport, but also to produce NO from NO2–, which is an unprecedented feature among the ferriheme proteins. The NPs were thus termed nitrite dismutase and classified as the novel group of enzymes EC 1.7.6.1 by the International Union of Biochemistry and Molecular Biology (IUBMB). To understand this novel catalytic reaction mechanism we recorded the X-ray structure of the protein to see how the nitrite ligand binds on the heme iron. It turned out that NPs bind the nitrite ligand via its nitrogen atom. This is in contrast to the heme proteins hemoglobin and myoglobin, that both bind the nitrite via its O atom. Based on this structure selected mutants of the protein were designed and analyzed by X-ray crystallography and spectroscopic methods. The results provide important insight into the catalytic mechanism of the nitrite dismutase reaction. The mutant L130R of the isoform NP4 is particularly interesting because it completely lost the enzymatic activity. Surprisingly, in the X-ray structure of NP4(L130R) without nitrite coordination, the R130 guanidine group appeared bound to the heme iron. This happens only at low temperatures and upon photon reduction of the heme iron, which is the case when the X- ray structure is recorded. However, guanidine bound metal complexes are generally rare in both biochemistry and synthetic chemistry and NP4(L130R) represents the first example of a metal porphyrin with guanidine coordination. This is surprising to happen in a protein and is an example of the extraordinary environments that protein pockets are capable to provide. Another part of the project dealt with the interaction between NP4 and thiol compounds like cysteine and homocysteine that occur in human blood. This study bases on previous reports that suggested that NO release is enhanced by these thiols. However, the previous study suggested that the thiols reduce the heme iron and tend to destroy the protein. In this study we showed that the protein forms stable complexes with the protein without iron reduction when the reaction is performed under oxygen free conditions. The X-ray structures of four such complexes were solved and spectroscopically characterized. Advanced EPR spectroscopy (ENDOR spectroscopy) allowed the detection of protonation at the thiol sulfur which settles a long-lasting discussion that this actually can happen in complex with heme proteins. NP7 is a special isoform because it can attach to negatively charged cell surfaces and thus deliver NO very efficient to a target cell. We now succeeded in the crystallization of NP7. For this purpose conditions had to be found that compensate the highly positive surface charge of this molecule. X-ray structures were solved with several ligands bound and of selected NP7 mutants. These structures can now be used as a basis to understand the molecular mechanisms of the NP7-membrane interaction. Overall, the results represent an important step for the understanding of NP structure and reactivity which is necessary for the development of pharmaceutical NO donor compounds.
Projektbezogene Publikationen (Auswahl)
- Guanidine–ferroheme coordination in the mutant protein nitrophorin 4(L130R), Angew. Chem. Int. Ed. 2012, 51, 4470- 4473
Chunmao He, Martin R. Fuchs, Hideaki Ogata, and Markus Knipp
(Siehe online unter https://doi.org/10.1002/anie.201108691) - Complexes of ferriheme nitrophorin 4 with low-molecular weight thiol(ate)s occurring in blood plasma, J. Inorg. Biochem.
Chunmao He, Koji Nishikawa, Özlen F. Erdem, Edward Reijerse, Hideaki Ogata, Wolfgang Lubitz, and Markus Knipp
(Siehe online unter https://doi.org/10.1016/j.jinorgbio.2013.01.012)
