International Collaboration in Chemistry: Protein Dynamics and Heme Protein Function
Zusammenfassung der Projektergebnisse
Heme proteins can perform a diverse variety of tasks, including electron transfer, storage and transport of small molecules, enzymatic functions and gas sensing, owing to the remarkable chemical properties of the heme prosthetic group. In this project, we have used time-resolved and steady state spectroscopy with monitoring in the UV/visible and infrared regions to investigate biochemical and structural properties of different heme proteins and to reveal differences and similarities in their behavior. Myoglobin (Mb) is the paradigm of an oxygen storage protein. The ligand binds covalently at the heme prosthetic group. Several transient ligand docking sites are located throughout the Mb matrix. They mediate ligand entry and escape. CuBMb is a copper-binding Mb double mutant (L29H–F43H) that was originally engineered to mimic the catalytic site of heme–copper oxidases. In copper-free CuBMb, the transient docking sites were accessible to the CO ligand upon photodissociation. In copper-bound CuBMb, however, ligands rather coordinated to the Cu ion. Ligands entering the heme pocket from the outside that normally would not be captured efficiently by the tight distal pocket housing the two additional large imidazole rings, bound at the Cu ion, i.e., the ion ensured efficient ligand trapping in CuBMb. The Cu ion also restricted the motions of the His64 side chain, which is the entry/exit door for ligand movement into the active site, which resulted in enhanced direct (geminate) and slow bimolecular CO rebinding. Cytochrome c (Cyt c) is a small globular heme protein involved in electron transfer. Therefore, the heme environment is rather rigid so as to keep the reorganization energy small. The protein matrix is dense, without any open volumes, and the heme iron is hexacoordinated. To imprint a ligand binding ability into Cyt c, the axial Met80 ligand was removed by site-directed mutagenesis. As expected, ligand binding was extremely fast even at very low temperatures because there are no residues hindering ligand access to the heme iron and there are no docking sites, where the ligands can reside without bond formation at the heme iron. In neuroglobin, the endogenous His64 side chain and the exogenous ligand compete for the sixth coordination site at the heme iron. We showed that heme movements govern the probability of the system to bind His64 or CO, that is, to select different binding pathways. Our main attention was focused on the physiologically important heme enzymes indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO) and inducible nitric oxide synthase (iNOS). Both hIDO and hTDO catalyze the oxidative cleavage of the L-Trp indole ring. We could show that formation of the ternary complex between the protein, the L-Trp substrate and the ligand is very different for the two enzymes. In hIDO, the ligand has to bind first. If the substrate binds first, the enzyme is shelved in a nonproductive state. It can only become catalytically active again after the substrate has vacated the active site. In contrast, in hTDO, a bound substrate favors ligand binding. In iNOS, the catalytic reaction can only occur if the protein has formed a tight dimer. Binding of both the L-Arg substrate and the H4B cofactor favors dimerization; both components are required to obtain the catalytically active complex that generates nitric oxide. We have studied NO binding and migration in iNOS and revealed that NO does not reside in the protein interior in a well-defined location or orientation, which may be favorable for NO release from the enzyme during catalysis. Interestingly, if NO is bound to a ferrous heme iron, light absorption causes preferential cleavage of the bond to the proximal thiolate; the Fe^II-NO bond is not affected.
Projektbezogene Publikationen (Auswahl)
- (2011) Ligand Migration in Human Indoleamine-2,3 Dioxygenase, IUBMB Life 63, 153-159
Nienhaus, K., Nickel, E., Lu, C. Y., Yeh, S. R., and Nienhaus, G. U.
- (2012) Ligand Binding to Heme Proteins: A Comparison of Cytochrome c Variants with Globins, J. Phys. Chem. B 116, 12180-12188
Nienhaus, K., Zosel, F., and Nienhaus, G. U.
(Siehe online unter https://doi.org/10.1021/jp306775n) - (2013) An engineered heme-copper center in myoglobin: CO migration and binding, Biochim. Biophys. Acta 1834, 1824-1831
Nienhaus, K., Olson, J. S., and Nienhaus, G. U.
(Siehe online unter https://doi.org/10.1016/j.bbapap.2013.02.031) - (2013) Reaction-Pathway Selection in the Structural Dynamics of a Heme Protein, Chemistry-A European Journal 19, 3558-3562
Nienhaus, K., Lutz, S., Meuwly, M., and Nienhaus, G. U.
(Siehe online unter https://doi.org/10.1002/chem.201203558) - (2014) Fourier Transform Infrared Spectroscopy Study of Ligand Photodissociation and Migration in Inducible Nitric Oxide Synthase, F1000Research 3, 290 (216 pages)
Horn, M., Nienhaus, K., and Nienhaus, G. U.
(Siehe online unter https://doi.org/10.12688/f1000research.5836.1) - (2014) Substrate Inhibition in Human Indoleamine 2,3-Dioxygenase, J. Phys. Chem. Lett. 5, 756-761
Weber, B., Nickel, E., Horn, M., Nienhaus, K., and Nienhaus, G. U.
(Siehe online unter https://doi.org/10.1021/jz500220k)
