MATURATION, STRUCTURE, AND ELECTRON TRANSFER PROPERTIES OF CYTOCHROME C

Cytochromes are heme containing proteins, which carry out diverse physiological functions, such as electron transfer, an important process in the energy metabolism of living cells. C type cytochromes are distinguished from other cytochromes by the covalent attachment of the heme group to the protein. The advantages of this covalent binding are so far unclear. The maturation of mitochondrial cytochrome c, i.e. the covalent binding of the heme cofactor, is catalysed by the enzyme cytochrome c heme lyase. Despite the importance and the widespread occurence of these proteins from yeast to human, we know little about the catalytic process or about the interaction of the two proteins and the heme. Recent studies have also shown that the mitochondrial cytochrome c has another fundamental role besides electron transfer in triggering the programmed cell death (apoptosis).

We express cytochrome c heme lyase in a bacterium and purify the protein. We study the interaction of the purified heme lyase, the heme and the apocytochrome (cytochrome c without heme) in order to clarify the details of cytochrome c maturation on a molecular level. According to our hypothesis the structure of both the heme lyase and the cytochrome may alter as a result of their interaction with each other and with the heme. Indirect information about this interaction is supplied by our observation of spontaneous cytochrome c maturation (covalent heme attachment in the absence of the cytochrome c heme lyase) at a much lower efficiency than in the presence of the heme lyase. However, the spontaneously matured cytochrome c has slightly different physico-chemical parameters than the native protein. This indicates that the function of the heme lyase is not only the catalysis (acceleration) of the covalent heme attachment, but also to facilitate the formation of the final, native conformation (structure) of cytochrome c. Without the heme lyase the first process can take place (albeit slowly, with low efficiency), but the second one cannot.

Cytochrome c is but one component of a complex energy transducing and storing apparatus consisting of various proteins and membranes. Metabolism is based on the migration of electrons through well organized protein chains towards lower energy, analogously to the current of electrons in the electric circuit from one pole through a load to the other pole. There is, however, a basic difference between the mechanism of electronic conductance in a metal wire and in a protein. Our goal is to better understand the mechanism of electronic conductance in proteins, and to study whether or not nature has optimized (and how) the conductance in certain important proteins. Our experiments are mainly performed on cytochrome c. We label the surface of the cytochrome with a molecule which becomes an electron source after its irradiation with a short laser pulse, and which donates its electron to the heme group within the protein, and recovers the electron afterwards. We can measure the rate of electron transfer and thereby compare various regions on the protein surface as well as various prospective routes (directions) within the protein in terms of the electronic conductance. We attempt to explain the link between the efficiency of electronic conductance and the structure of the protein with model calculations. In the figure we have colored the surface of cytochrome c according to the calculated efficiency of electron transfer from the heme (in the middle) to the various regions of the surface as green, red, and blue for average, good, and poor conductance, respectively.

Further research plans, possible utilization of the results

We plan to investigate the electron transfer routes, the efficiency of electron (and charge) transfer and its energetic utilization in the more complex but similarly important physiological oxidizing partner of cytochrome c, the cytochrome c oxidase protein. Cytochrome c itself is a promising candidate as a component of biomolecule-based sensors, bioelectronic designs, so the understanding of its electric conductance is a high priority. Within the framework of an international collaboration we also plan to incorporate cytochrome c into hybrid biophotonic architectures – photonic crystals based on porous silicon. Thereby we expect to tune the optical properties of the semiconductor-based photonic crystals using the colored cytochrome protein. Conversely, we intend to study the effects of the interesting nonlinear optical phenomena characteristic of the photonic crystals on the optical properties of the protein.