Kinetics and Energetics of Intramolecular Electron Transfer in Single-Point Labeled TUPS-Cytochrome c Derivatives.

Electron transfer within and between proteins is a fundamental biological phenomenon, in which efficiency depends on several physical parameters. We have engineered a number of horse heart cytochrome c single-point mutants with cysteine substitutions at various positions of the protein surface. To these cysteines, as well as to several native lysine side chains, the photoinduced redox label 8-thiouredopyrene-1,3,6-trisulfonate (TUPS) was covalently attached. The long-lived, low potential triplet excited state of TUPS, generated with high quantum efficiency, serves as an electron donor to the oxidized heme c. The rates of the forward (from the label to the heme) and the reverse (from the reduced heme back to the oxidized label) electron transfer reactions were obtained from multichannel and single wavelength flash photolysis absorption kinetic experiments. The electronic coupling term and the reorganization energy for electron transfer in this system were estimated from temperature-dependent experiments and compared with calculated parameters using the crystal and the solution NMR structure of the protein. These results together with the observation of multiexponential kinetics strongly support earlier conclusions that the flexible arm connecting TUPS to the protein allows several shortcut routes for the electron involving through space jumps between the label and the protein surface.

Disentangling electron tunneling and protein dynamics of cytochrome c through a rationally designed surface mutation.

Nonexponential distance dependence of the apparent electron-transfer (ET) rate has been reported for a variety of redox proteins immobilized on biocompatible electrodes, thus posing a physicochemical challenge of possible physiological relevance. We have recently proposed that this behavior may arise not only from the structural and dynamical complexity of the redox proteins but also from their interplay with strong electric fields present in the experimental setups and in vivo (J. Am Chem. Soc. 2010, 132, 5769-5778). Therefore, protein dynamics are finely controlled by the energetics of both specific contacts and the interaction between the protein's dipole moment and the interfacial electric fields. In turn, protein dynamics may govern electron-transfer kinetics through reorientation from low to high donor-acceptor electronic coupling orientations. Here we present a combined computational and experimental study of WT cytochrome c and the surface mutant K87C adsorbed on electrodes coated with self-assembled monolayers (SAMs) of varying thickness (i.e., variable strength of the interfacial electric field). Replacement of the positively charged K87 by a neutral amino acid allowed us to disentangle protein dynamics and electron tunneling from the reaction kinetics and to rationalize the anomalous distance dependence in terms of (at least) two populations of distinct average electronic couplings. Thus, it was possible to recover the exponential distance dependence expected from ET theory. These results pave the way for gaining further insight into the parameters that control protein electron transfer.

Porous silicon/photosynthetic reaction center hybrid nanostructure.

The purified photosynthetic reaction center protein (RC) from Rhodobacter sphaeroides R-26 purple bacteria was bound to porous silicon microcavities (PSiMc) either through silane-glutaraldehyde (GTA) chemistry or via a noncovalent peptide cross-linker. The characteristic resonance mode in the microcavity reflectivity spectrum red shifted by several nanometers upon RC binding, indicating the protein infiltration into the porous silicon (PSi) photonic structure. Flash photolysis experiments confirmed the photochemical activity of RC after its binding to the solid substrate. The kinetic components of the intraprotein charge recombination were considerably faster (τ(fast) = 14 (±9) ms, τ(slow) = 230 (±28) ms with the RC bound through the GTA cross-linker and only τ(fast) = 27 (±3) ms through peptide coating) than in solution (τ(fast) = 120 (±3) ms, τ(slow) = 1387 (±2) ms), indicating the effect of the PSi surface on the light-induced electron transfer in the protein. The PSi/RC complex was found to oxidize the externally added electron donor, mammalian cytochrome c, and the cytochrome oxidation was blocked by the competitive RC inhibitor, terbutryne. This fact indicates that the specific surface binding sites on the PSi-bound RC are still accessible to external cofactors and an electronic interaction with redox components in the aqueous environment is possible. This new type of biophotonic material is considered to be an excellent model for new generation applications at the interface of silicon-based electronics and biological redox systems designed by nature.

Mapping local electric fields in proteins at biomimetic interfaces.

We present a novel approach for determining the strength of the electric field experienced by proteins immobilised on membrane models. It is based on the vibrational Stark effect of a nitrile label introduced at different positions on engineered proteins and monitored by surface enhanced infrared absorption spectroscopy.

Maturation of a eukaryotic cytochrome c in the cytoplasm of Escherichia coli without the assistance by a dedicated biogenesis apparatus.

Maturation of c-type cytochromes involves the covalent and stereospecific enzymatic attachment of a heme b via thioether linkages to two conserved cysteines within apocytochromes. Horse cytochrome c is readily matured into its native holoform in the cytoplasm of E. coli when co-expressed with yeast cytochrome c heme lyase. Here we report the low yield formation of holocytochrome with covalently attached heme also in the absence of heme lyase. This is the first demonstration of in vivo maturation of a eukaryotic cytochrome c in a prokaryotic cytoplasm without the assistance by a dedicated enzymatic maturation system. The assembled cytochrome c can be oxidized by cytochrome c oxidase, indicating the formation of a functional protein. The absorption spectrum is typical of a low spin, six coordinated c-type heme. Nevertheless, minor spectral differences relative to the native cytochrome c, deviation of the midpoint reduction potential and slightly altered kinetic parameters of the interaction with cytochrome c oxidase emphasize the importance of cytochrome c heme lyase in folding cytochrome c into its native conformation.

Improved system for heterologous expression of cytochrome c mutants in Escherichia coli.

We improved an already existing cytochrome c expression system to a reliable, tightly controllable one to achieve a higher expression yield for single cysteine mutants of horse cytochrome c. The protein is heterologously overexpressed in E. coli together with the maturation coordinating enzyme heme lyase from yeast. Various plasmid constructs and host strains were tested for protein expression yield and routinely around 35 mg/L yield was achieved, which is a good result for a post-translationally modified enzyme. The purpose of producing cysteine mutants is to position accessible cysteine residues on the surface of cytochrome c which can be labeled with a photoactive redox dye, 8-thiouredopyrene-1,3,6-trisulfonate, TUPS. TUPS labeled proteins have been used for intramolecular and intermolecular electron transfer measurements. Here, we initiate the photoreduction of cytochrome c oxidase, the natural electron acceptor partner of cytochrome c by an appropriate cytochrome c mutant labeled with TUPS. The electron transfer from cytochrome c to the first cytochrome oxidase redox cofactor, copper A, is shown to be very fast.

Complex kinetics of the electron transfer between the photoactive redox label TUPS and the heme of cytochrome c.

The photoinduced covalent redox label 8-thiouredopyrene-1,3,6-trisulfonate (TUPS) has been attached to two lysine residues (K8 and K39) at opposite sides of horse heart cytochrome c, as well as to cysteines, at the same positions, introduced by site-directed mutagenesis. Electron transfer between TUPS and the heme of cytochrome c deviates from the expected monoexponential kinetic behavior. Neither the overall rate nor the individual exponential components of electron transfer, as followed by kinetic absorption spectroscopy, correlate with the length of the covalent link connecting the dye with the protein. Molecular dynamics calculations show that TUPS can approach the protein surface and occupy several such positions. This heterogeneity may explain the multiexponential electron-transfer kinetics. The calculated optimal electron-transfer pathways do not follow the covalent link but involve through space jumps from the dye to the protein moiety, effectively decoupling the length of the covalent link and the electron-transfer rates.

Redox photochemistry of thiouredopyrenetrisulfonate.

1-Thiouredopyrene-3,6,8-trisulfonate (TUPS) has recently been used as a photoinduced covalent redox label capable of reducing various cofactors of proteins. A new reaction of this dye, whereby its excited triplet state oxidizes suitable electron donors, is now reported. The characteristic difference spectrum of the reduced radical of TUPS is determined. We also observe the self-exchange electron transfer between two TUPS molecules in their triplet excited states and determine the reaction scheme and the rate constants of the various pathways in the process of triplet depletion. The ability of photoexcited TUPS to withdraw an electron from reduced cytochrome-c is also observed. It is thus demonstrated that TUPS is an appropriate photoinduced covalent redox label for initiating both the oxidative and reductive phases of electron transfer processes in biological macromolecules.