Remarkably high activities of testicular cytochrome c in destroying reactive oxygen species and in triggering apoptosis.

Hydrogen peroxide (H(2)O(2)) is the major reactive oxygen species (ROS) produced in sperm. High concentrations of H(2)O(2) in sperm induce nuclear DNA fragmentation and lipid peroxidation and result in cell death. The respiratory chain of the mitochondrion is one of the most productive ROS generating systems in sperm, and thus the destruction of ROS in mitochondria is critical for the cell. It was recently reported that H(2)O(2) generated by the respiratory chain of the mitochondrion can be efficiently destroyed by the cytochrome c-mediated electron-leak pathway where the electron of ferrocytochrome c migrates directly to H(2)O(2) instead of to cytochrome c oxidase. In our studies, we found that mouse testis-specific cytochrome c (T-Cc) can catalyze the reduction of H(2)O(2) three times faster than its counterpart in somatic cells (S-Cc) and that the T-Cc heme has the greater resistance to being degraded by H(2)O(2). Together, these findings strongly imply that T-Cc can protect sperm from the damages caused by H(2)O(2). Moreover, the apoptotic activity of T-Cc is three to five times greater than that of S-Cc in a well established apoptosis measurement system using Xenopus egg extract. The dramatically stronger apoptotic activity of T-Cc might be important for the suicide of male germ cells, considered a physiological mechanism that regulates the number of sperm produced and eliminates those with damaged DNA. Thus, it is very likely that T-Cc has evolved to guarantee the biological integrity of sperm produced in mammalian testis.

The redox couple of the cytochrome c cyanide complex: the contribution of heme iron ligation to the structural stability, chemical reactivity, and physiological behavior of horse cytochrome c.

Contrary to most heme proteins, ferrous cytochrome c does not bind ligands such as cyanide and CO. In order to quantify this observation, the redox potential of the ferric/ferrous cytochrome c-cyanide redox couple was determined for the first time by cyclic voltammetry. Its E0' was -240 mV versus SHE, equivalent to -23.2 kJ/mol. The entropy of reaction for the reduction of the cyanide complex was also determined. From a thermodynamic cycle that included this new value for the cyt c cyanide complex E0', the binding constant of cyanide to the reduced protein was estimated to be 4.7 x 10(-3) L M(-1) or 13.4 kJ/mol (3.2 kcal/mol), which is 48.1 kJ/mol (11.5 kcal/mol) less favorable than the binding of cyanide to ferricytochrome c. For coordination of cyanide to ferrocytochrome c, the entropy change was earlier experimentally evaluated as 92.4 J mol(-1) K(-1) (22.1 e.u.) at 25 K, and the enthalpy change for the same net reaction was calculated to be 41.0 kJ/mol (9.8 kcal/mol). By taking these results into account, it was discovered that the major obstacle to cyanide coordination to ferrocytochrome c is enthalpic, due to the greater compactness of the reduced molecule or, alternatively, to a lower rate of conformational fluctuation caused by solvation, electrostatic, and structural factors. The biophysical consequences of the large difference in the stabilities of the closed crevice structures are discussed.

Intermolecular biological electron transfer: an electrochemical approach.

We investigated the electron transfer (ET) rates between a well-defined gold electrode and cytochrome c immobilized at the carboxylic acid terminus of alkanethiol self-assembled monolayers (SAMs) by using the potential modulated electroreflectance technique. A logarithmic plot of ET rates against the chain length of the alkanethiol is linear with long chain alkanethiols. The ET rates become independent of the chain length with short alkanethiols. It is proposed that the rate-limiting ET step through short alkyl chains results from a configurational rearrangement process preceding the ET event. This "gating" process arises from a rearrangement of the cytochrome c from a thermodynamically stable binding form on the carboxylic acid terminus to a configuration, which facilitates the most efficient ET pathways (surface diffusion process). We propose that the lysine-13 of mammalian cytochrome c facilitates the most efficient ET pathway to the carboxylate terminus and this proposal is supported by the ET reaction rate of a rat cytochrome c mutant (RC9-K13A) [Elektrokhimiya (2001) in press], in which lysine-13 is replaced by alanine. The ET rate of K13A is more than six orders of magnitude smaller than that of the native protein.