cytochrome-c-t and zinc-hematoporphyrin

Multifunctional gold nanoparticles (AuNPs) are of great interest, owing to their vast potential for use in many areas including sensing, imaging, delivery, and medicine. A key factor in determining the biological activity of multifunctional AuNPs is the quantification of surface conjugated molecules. There has been a lack of accurate methods to determine this for multifunctionalized AuNPs. We address this limitation by using a new method based on the deconvolution and Levenberg-Marquardt algorithm fitting of UV-visible absorption spectrum to calculate the precise concentration and number of cytochrome

Topics: Cytochromes c; Dynamic Light Scattering; Gold; Metal Nanoparticles; Multifunctional Nanoparticles; Spectrum Analysis

An artificial zinc porphyrin-myoglobin-based photo-chemical energy conversion system, consisting of ZnPP-Mb or ZnPE(1)-Mb as a photosensitizer, NADP(+) as an electron acceptor, and triethanolamine as an electron donor, has been constructed to mimic photosystem I. The photoirradiated product is able to reduce a single-electron acceptor protein cytochrome c, but cannot catalyze the two-electron reduction of acetaldehyde by alcohol dehydrogenase, thus demonstrating a single electron transfer mechanism. Furthermore, the artificial system can bifunctionally promote oxidoredox reactions, depending on the presence or absence of a sacrificial electron donor, thus suggesting its potential application in electrochemical regeneration steps involved in chemical transformation and/or energy conversion.

Topics: Cytochromes c; Ethanolamines; Fluorescence Polarization; Metalloporphyrins; Models, Chemical; Molecular Structure; Myoglobin; NADP; Oxidation-Reduction; Photochemical Processes; Photosensitizing Agents; Photosynthesis

We demonstrate that Zn(II) porphyrin in Zn(II)cytochrome c (Zn cyt c) is a fluorescence resonance energy transfer (FRET) donor to an Alexa660 dye acceptor. The energy transfer efficiency is dependent on the distance between the two fluorophores as shown through protein denaturation studies of five Zn cyt c variants labeled with Alexa660 in different positions. The relative quantum yield, excitation and emission energies, and labeling efficiencies of this donor-acceptor pair allow for a method of analysis based on sensitized emission of the acceptor. These studies show that Zn(II) porphyrin is an effective energy donor for measurement of molecular-scale distances by FRET.

Topics: Crystallography, X-Ray; Cytochromes c; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Metalloporphyrins; Models, Molecular; Protein Conformation; Protein Folding; Zinc

FRET between the zinc porphyrin (ZnP) chromophore in zinc-substituted cytochrome c (Zn-cyt c) and an Alexa Fluor dye attached to specific surface sites was used to characterize Zn-cyt c unfolding. The use of ZnP as a fluorescent acceptor eliminates the need to doubly label the protein with exogenous dyes to perform FRET experiments in which both donor and acceptor fluorescence is monitored. The requirement for attachment of only one dye also minimizes perturbation to the protein. This sensitive technique allowed for the determination of distances between the label placed at six different sites and ZnP through a range of denaturant concentrations. Fitting of the data to a three-state model provides distances in the unfolding intermediate. The use of ZnP as a fluorescent acceptor of energy in FRET has a significant potential for application to a range of other systems including heme-binding proteins and proteins to which a covalently attached heme tag may be added.

Topics: Cytochromes c; Fluorescence Resonance Energy Transfer; Metalloporphyrins; Models, Molecular; Protein Folding; Zinc

The kinetics of electron transfer from the triplet-excited Zn-porphyrin to a Ru(NH(3))(5)(His-33)(3+) complex have been measured in Zn-substituted ruthenium-modified cytochrome c under denaturing conditions. In the folded protein, the electron-tunneling rate constant is 7.5 x 10(5) s(-1). As the protein is denatured with guanidine hydrochloride, a faster adiabatic electron-transfer reaction appears (4.0 x 10(6) s(-1), [guanidine hydrochloride] = 5.4 M) that is limited by the rate of intrachain diffusion to bring the Zn-porphyrin and Ru complex into contact. The 250-ns contact time for formation of a 15-residue loop in denatured cytochrome c is in accord with a statistical model developed by Camacho and Thirumalai [Camacho, C. J. & Thirumalai, D. (1995) Proc. Natl. Acad. Sci. USA 92, 1277-1281] that predicts that the most probable transient loops formed in denatured proteins are comprised of 10 amino acids. Extrapolation of the cytochrome c contact time to a 10-residue loop sets the folding speed limit at approximately 10(7) s(-1).

Topics: Cytochrome c Group; Cytochromes c; Electron Transport; Guanidine; Histidine; Kinetics; Macromolecular Substances; Metalloporphyrins; Models, Molecular; Organometallic Compounds; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Tertiary; Ruthenium; Sucrose; Time Factors; Zinc