Text

theses

ETD_4164

Ph.D.

Includes bibliographical references

Includes vita

by Katarzyna Izabela Jankowska

http://hdl.rutgers.edu/1782.1/rucore10002600001.ETD.000066553

doi:10.7282/T3M907FH

ETD doctoral

Electron transfer (ET) reactions play a crucial role in biological systems. They occur at critical steps of numerous metabolic pathways and are studied in a variety of ways. One of the methods involves photoinduced ET investigation of chemically modified proteins, which are labeled or transformedto be photoactive. The most popular class of redox active proteins which allow modifications facilitating the study of ET reactions are heme proteins such as cytochromes. Their properties include colored prosthetic groups, varied oxidation states, and diverse biological functions which have provided a rich and fertile ground for study by chemists, biophysicists and biologists. The native iron-containing heme dissipates the excitation energy through rapid radiationless transitions which lead to extremely short excited state lifetimes in the femtosecond range. The replacement of iron by diamagnetic zinc at the porphyrin center dramatically changes the photophysics of the enzyme. Proteins containing zinc porphyrins exhibit both fluorescence and long lived phosphorescence Thanks to their emissive nature, they are amenable to highly sensitive single photon counting techniques, which permit kinetic studies over a very broad range of concentrations and time scales.

A large part of the research reported here has focused on examining intermolecular interactions in zinc substituted heme proteins probed by phosphorescence quenching. First, photoinduced ET reactions between zinc substituted cytochrome P450cam (ZnP450) and small organic compounds capable of accessing the protein’s hydrophobic channel and binding close to active site in a fashion that mimics its native substrate, camphor, were investigated.The heme – to - zinc protoporphyrin exchange revealed the existence of two conformers of the substituted protein (F420 and F450) which exhibited different photochemical and photophysical properties. The ET behavior of form F420 suggests that hydrophobic redox-active ligands are able to penetrate the hydrophobic channel and locate themselves in the direct vicinity of the Zn-porphyrin. In contrast, the slower ET quenching rates observed in the case of F450 indicate that the association is weak and occurs outside of the protein channel. Therefore, we conclude that form F420 corresponds to the open structure of the native cytochrome P450cam, while form F450 has a closed or partially closed channel that is characteristic of the camphor-containing cytochrome P450cam.

Both forms of ZnP450 were examined in the presence of ligands possessing long aliphaticchains to explore electron and energy transfer processes in those systems. The triplet state lifetime of form F420 decreases in all studied cases, while the emission of 3F450 remains unchanged in the presence of the selected ligands. The ET and TT rates obtained from 3F420 quenching allowed the calculation of the separation between the Zn-center and quenchers. The estimated D-A distances are consistent with those previously
obtained from X-ray structures of native cytochrome P450 and imply ligation close to the active center. Moreover, similarly as in native P450, quenchers with a hydrophobic tether induce conformational changes in ZnP450. These data show that ZnP450 mimics the behavior of the native enzyme and can be used to study protein - ligand and protein -protein interactions.

In the second part of this thesis a water soluble octacarboxyhemicarcerand was used as a shuttle to transport redox–active substrates across the aqueous medium and deliver them to the target protein. Hydrophobic electron donors and acceptors wereencapsulated within the hemicarcerand, and photoinduced electron transfer between the Zn-substitutedcytochrome c and the host-guest complexes was used to probe the association between the negatively charged hemicarceplex and the positively charged protein. ET mediated by the protein-bound hemicarcerand is much faster than that due to diffusional encounters with the respective free donor or acceptor in solution. The results show that the hemicarcerand is capable of exhibiting ‘induced fit’ behavior characteristic of protein-protein interactions. The kinetic behavior of these systems depends on the relative strength of the protein-hemicarcerand and guest-hemicarcerand interactions. When Kencaps >> Kassoc, the hemicarcerand transports the ligand to the protein while protecting it from the aqueous medium. But if Kassoc > Kencaps the docked cage can act as an artificial receptor.

In addition, the water soluble Cram-type hemicarcerand has been used as a gas capture device. This project involved encapsulation studies of hydrophobic gases such as sulfur hexafluoride or butane. The interactions between the guest and the host were probed by 1D relaxation and 2D NOESY experiments. The host:guest complex in a ratio of 1:1 was the dominant form for both studied gases, however additional NMR signals in the case of butane suggest imprisonment of two guests. The encapsulation of the second molecule is possible thanks to structural flexibility of a hemicarcerand, which can adapt to the guest. Furthermore, the fundamental question of the gas-like vs. liquid like behavior of the guest(s) in the inner phase environment of the hemicarcerand has been addressed. Our observations imply liquid like behavior for the 1:1 host: guest complex and solid-like behavior for the 1:2 host: butane complex, thus agreeing with Cram’s conjecture that the cage interior can be designed to be vacuum-like, liquid-like, or even solid-like depending on the fraction of the space occupied by the guest(s).

The presented work not only contributes new knowledge about photoinduced electron and energy transfer reactions in proteins but also introduces interesting materials for bio-sensing, biomedical or biocatalytic applications.

The author owns the copyright to this work.

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