Cytochrome diffusion constant

The molecular weight of cytochrome c peroxidase has been determined to be 34,100 on the basis of a sedimentation constant of 3.55 S, a diffusion constant of 9.44 F, and a partial specific volume of 0.733 ml/g (4 )-The enzyme exists as a monodisperse monomer containing one ferric protoporphyrin IX, which is noncovalently bound (/, , 14). No other transition metal is detected in crystalline preparations of the enzyme (22). The apoenzyme moiety is an acidic protein with an isoelectric point at pH... 

Perhaps the strongest evidence in favor of the theory that the cytochrome oxidase system is composed of a single protein comes from the physicochemical studies of Takemori. Purified cytochrome oxidase appears in the ultracentrifuge as a nondispersed protein (sedimentation constant = 21.9 diffusion constant =3.58 x 10 partial specific volume = 0.72). The molecular weight of the protein was calculated on the basis of these studies and was found to equal 530,000. 

A great role in substantiating the importance of electron tunneling reactions was played by the work of De Vault and Chance in 1966 where the characteristic time, t1/z, of electron transfer from the heme site of the cytochrome molecule to the chlorophyll molecule in a bacterium was shown to be constant within the temperature range of 130 to 4.2 K [4]. The temperature independence of t1/2 permitted one to reject a diffusion mechanism for the process. However, it was still impossible to exclude the possibility of the reaction to proceeding via direct contact between the active sites of the reacting molecules. 

The opportunity of obtaining direct electrochemistry of cytochrome c and other metalloproteins at various electrode materials such as modified gold and pyrolytic graphite has led to numerous reports of heterogeneous electron transfer rates and mechanisms between the protein and the electrode. In all the reports, Nicholson s method (37) was employed to calculate rate constants, which were typically within the range of 10" -10 cm sec with scan rates varying between 1 and 500 mV sec This method is based on a macroscopic model of the electrode surface that assumes that mass transport of redox-active species to and from the electrode occurs via linear diffusion to a planar disk electrode and that the entire surface is uniformly electroactive, i.e., the heterogeneous electron transfer reaction can take place at any area. 

Figure 11. Relaxation, 4>(r), of the center of energy is plotted for wave packets propagated by the normal modes of cytochrome c hydrated by 400 water molecules (circles) and myoglobin (squares). Curve is a stretched exponential, Eq. (33), with p = 2v = 0.52, the value fit to the computed energy diffusion data for cytochrome c plotted in Fig. 10, and time constant, t — 11 ps.

Recently it was proposed that the apparently slow heterogeneous electron-transfer rates for such proteins as cytochrome c, cytochrome b5, plasto-cyanin, and ferredoxin are an artifact of the experimental approach (25). Instead of assuming that protein molecules react at a planar and essentially homogeneous surface, it is assumed instead that movement of the protein occurs predominantly by radial diffusion to very small, specific sites. These sites are presumed to facilitate very rapid electron transfer at the reversible potential while the rest of the surface remains inactive. Thus, the modified electrode surface behaves like an array of microelectrodes. If this theory is used to treat previous data, much higher electron-transfer rate constants are obtained. Although this theory deserves more detailed scrutiny, it may serve... 

Using the methylviologen cation radical (MV +) formed by pulse radiolysis, monophasic kinetics of cytochrome reduction are observed with a rate constant of 4.5 X 108 M 1/s (1.1 X 108 M 1/s on a per heme basis) at pH 8.0 with the Hildenborough cytochrome (36). This very fast second-order process approaches the diffusion controlled limit. Moreover, the reverse reaction can be estimated to be 7.8 X 104 M-1/s, which suggests that the reaction takes place primarily with the highest potential heme (the A E 0 between heme I and MV + is 190 mV, consistent with an equilibrium constant of approximately 103). Interestingly, the kinetics with MV + are ionic strength dependent, which is consistent with a plus-plus interaction,... 

X 10 M" s . This value is equal to that calculated theoretically for a diffusion controlled reaction occurring when collisions are at high frequencies. This is not the case for the cytochrome hi core - cytochrome c complex since the affinity of the latter complex is found equal to 2.5 pM while the dissociation constant of the flavocytochrome hi - cytochrome c complex is equal to 0.1 pM. 

Figure 18. (A) Cyclic voltammetry of purified cytochrome c at doped indium oxide optically transparent electrodes. Solution contained 73 /uiM cytochrome c, 0.21 M Tris, 0.24 M cacodylic acid, pH 7.0, 0.20 M ionic strength. Electrode area = 0.71 cm. Potential scan rates in mV/s are (a) 100 (b) 50 (c) 20 (d) 10 (e) 5.0 (f) 2.0. (B) Derivative cyclic voltabsorptometry of purified cytochrome c at a tin-doped indium oxide optically transparent electrode. Same conditions as described above. Circles are calculated derivative cyclic voltabsorptometric responses for 73 /iM cytochrome c, formal potential = 0.260 V, n = 1.0, diffusion coefficient of oxidized and reduced cytochrome c = 1.2 x 10 cm /s, difference molar absorptivity at 416 nm = 57,000 cm" formal heterogeneous electron transfer rate constant = 1.0 x 10 cm/s, and electrochemical transfer coefficient = 0.5. Adapted from Reference (126) with permission.

A theoretical analysis of charge distribution within supercomplexes (or clusters in which the movement of diffusible carriers is restricted) has been developed by Lavergne et al [4]. This theory predicts the evolution of the redox state of the carriers under continuous illumination or flash excitation for any cluster stoichiometry. The predictive power of this treatment is illustrated by the analysis of the light-induced oxidation of primary and secondary donors in isolated centers of Rhodopseudomonas viridis (Fig. 3). In this case, it is definitely established that the secondary donors (cytochromes) are irreversibly bound to the reaction center. In the absence of mediators, no electron exchange is expected to occur between photocenters. In the presence of 200yM ascorbate, only two of the four cytochromes (cyt 556 and cyt 559) are in their reduced state prior to the illumination. As expected, the apparent equilibrium constant between P and the cytochromes measured during the course of illumination is much lower than that computed from the value of the redox potentials (K = 50 for cyt 559 and K 1500 for cyt 556). The fit between the experimental data and the theoretical simulation (dashed lines) is excellent and clearly demonstrates that the measurement of electron transfer reactions under weak illumination is a powerful tool to characterize the degree of structuration of a photosynthetic electron transfer chain. 

A number of questions remain concerning the dynamics of the protein-electrode interaction. Do experimental data give any idea about the rates, relative or otherwise, of the electron-transfer step The clearest result so far has been the determination of ke, for horse heart cytochrome c at 4,4 -bipyridyl-modified Au with the rotating ring-disk technique as mentioned above [65], There have been a number of determinations of compound heterogeneous rate constants for protein electrochemistry, mostly using Nicholson s method [124] for their estimation from CV peak separations. All calculations have assumed that the mass transport can be treated in terms of linear diffusion to a uniform planar electrode surface. Bond and co-workers have pointed out [125,126] that in many instances this is unlikely to be... 

Redox conduction via cytochromes is a thermally activated process. Conductivity decreases with a decrease in temperature, as the diffusion coefficient that comprises the rate constant, for the redox reactions is also temperature dependent and the rate decreases exponentially with a decrease in temperature [27] according to ... 

The formation of SOi in the reaction of reduced cytochrome P-450 with bisulfite in an aqueous solution near pH 7 has also been observed [26]. The reaction was very slow, with the E8R signal due to 802 increasing over a period of several hours. This reaction was probably not with H8O3", but rather with the small amount of free SO2 in the solution. This would be consistent with the observation that whereas 8O2 is reduced by the methylviologen radical anion with a near diffusion-controlled rate constant, 1.2 x 10 L mol" s", H8O3" reacts slowly, if at all with that radical anion [25]. 802" may also be an important intermediate leading to the formation of elemental sulfur in the radiolysis of aqueous 8O2 solutions [27]. 

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