The role of cytochrome c diffusion in mitochondrial electron transport.

We have compared the modes and rates of cytochrome c diffusion to the rates of cytochrome c-mediated electron transport in isolated inner membranes and in whole intact mitochondria. For inner membranes, an increasing ionic strength results in an increasing rate of cytochrome c diffusion, a decreasing concentration (affinity) of cytochrome c near the membrane surface as well as near its redox partners, and an increasing rate of electron transport. For intact mitochondria, an increasing ionic strength results in a parallel, increasing rate of cytochrome c-mediated electron transport. In both inner membranes and intact mitochondria the rate of cytochrome c-mediated electron transport is highest at physiological ionic strength (100-150 mM), where the diffusion rate of cytochrome c is highest and its diffusion mode is three-dimensional. In intact mitochondria, succinate and duroquinol-driven reduction of endogenous cytochrome c is greater than 95% at all ionic strengths, indicating that cytochrome c functions as a common pool irrespective of its diffusion mode. Using a new treatment to obtain bimolecular diffusion-controlled collision frequencies in a heterogenous diffusion system, where cytochrome c diffuses laterally, pseudo-laterally, or three-dimensionally while its redox partners diffuse laterally, we determined a high degree of collision efficiency (turnover/collisions) for cytochrome c with its redox partners for all diffusion modes of cytochrome c. At physiological ionic strength, the rapid diffusion of cytochrome c in three dimensions and its low concentration (affinity) near the surface of the inner membrane mediate the highest rate of electron transport through maximum collision efficiencies. These data reveal that the diffusion rate and concentration of cytochrome c near the surface of the inner membrane are rate-limiting for maximal (uncoupled) electron transport activity, approaching diffusion control.

The Role of Cytochrome c Diffusion in Mitochondrial Electron Transport* (Received for publication, March 27, 1987) Sharmila Shaila Gupte and Charles R. Hackenbrock We have compared the modes and rates of cytochrome c diffusion to the rates of cytochrome c-mediated electron transport in isolated inner membranes and in whole intact mitochondria. For inner membranes, an increasing ionic strength results in an increasing rate of cytochrome c diffusion, a decreasing concentration (affinity) of cytochrome c near the membrane surface as well as near its redox partners, and an increasing rate of electron transport. For intact mitochondria, an increasing ionic strength results in a parallel, increasing rate of cytochrome c-mediated electron transport. In both inner membranes and intact mitochondria the rate of cytochrome c-mediated electron transport is highest at physiological ionic strength (100-150 mM), where the diffusion rate of cytochrome c is highest and its diffusion mode is three-dimensional. In intact mitochondria, succinate and duroquinoldriven reduction of endogenous cytochrome c is >95% at all ionic strengths, indicating that cytochrome c functions as a common pool irrespective of its diffusion mode. Using a new treatment to obtain bimolecular diffusion-controlled collision frequencies in a heterogenous diffusion system, where cytochrome c diffuses laterally, pseudo-laterally, or three-dimensionally while its redox partners diffuse laterally, we determined a high degree of collision efficiency (turnover/ collisions) for cytochrome c with its redox partners for all diffusion modes of cytochrome c. At physiological ionic strength, the rapid diffusion of cytochrome c in three dimensions and its low concentration (affinity) near the surface of the inner membrane mediate the highest rate of electron transport through maximum collision efficiencies. These data reveal that the diffusion rate and concentration of cytochrome c near the surface of the inner membrane are rate-limiting for maximal (uncoupled) electron transport activity, approaching diffusion control.
Mitochondrial electron transport mediated by cytochrome c has been studied extensively (e.g. Refs. 1-6) and traditionally, cytochrome c and its redox partners have been viewed as a structurally stable macromolecular aggregate, comprising the heme protein portion of the electron transport "chain" (7,8). However, using fluorescence recovery after photobleaching (9) and immunofluorescence imaging (lo), we have determined that the electron transferring components of the mi-Grant GM28704 and National Science Foundation Grant PCM84-*This research was supported by National Institutes of Health 02569. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tochondrial inner membrane are independent lateral diffusants and, for a homogenous (two-dimensional) diffusion system, determined that ordered chains (7,8), respiratory assemblies (ll), or transient aggregates (12)(13)(14) of cytochrome c and its redox partners are not necessary for cytochrome c-mediated electron transport. We have concluded that the overall process of mitochondrial electron transport includes diffusion and random collisions of the redox components which represents the basis for the random collision model of electron transport (9).
In our preceding paper (15) we reported on the rates and modes (lateral, pseudo-lateral, and three-dimensional) of cytochrome c diffusion and calculated the collision frequencies of cytochrome c with its redox partners for each diffusion mode. In this paper, we compare the modes and rates of cytochrome c diffusion to the rates of electron transfer from cytochrome bc1 to cytochrome oxidase in isolated inner membranes and in whole intact mitochondria at various ionic strengths. Using the kinetic data from this paper and new treatment to obtain collision frequencies in heterogenous diffusion systems (15), we calculate here the collision efficiencies (turnover/collisions) of cytochrome c with its redox partners. The collision efficiencies reveal that the diffusion rate and concentration (affinity) of cytochrome c near the surface of the inner membrane are rate-limiting for maximum (uncoupled) electron transport activity, approaching diffusion control. At physiological ionic strength, cytochrome c diffuses rapidly in a three-dimensional mode and has the lowest affinity for the inner membrane while mediating the highest rate of electron transport through maximum collision efficiencies with its redox partners.
Enzyme Assays-Duroquinol oxidase activity was measured polarographically with a Clark oxygen electrode (17) according to Schneider et al. (18). For an individual set of experiments, electron transport activity at each ionic strength was measured in triplicate and all samples in an individual set were statistically randomized. Three different sets using different inner membrane preparations were used to determine the duroquinol oxidase activity at each ionic strength. Nonenzymatic rates of electron transport were measured in the presence of 0.2 Fg/ml antimycin A and 2 mM KCN added to the chamber prior to the addition of inner membranes. Eadie-Hofstee plots of duroquinol oxidase activity were analyzed using a twocomponent linear regression computer program. Succinate reductase and duroquinol reductase activities were measured using a SLM- The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; duroquinol, tetramethyl-p-benzoquinol.

5248
This is an Open Access article under the CC BY license.   Aminco DW-2C spectrophotometer in dual wavelength mode.
were purchased from Sigma. All chemicals were reagent grade.
Materials-Horse heart cytochrome c (type VI) and duroquinone

Characteristics of Cytochrome c-mediated Electron Transport in Isolated Inner
Membranes-Maximum cytochrome cmediated duroquinol oxidase activity and apparent K,,, values, as a measure of the affinity of cytochrome c for its redox partners, were determined for inner membranes from 0 to 150 mM ionic strengths as utilized in our diffusion measurements of cytochrome c (15). Eadie-Hofstee plots of duroquinol oxidase activity (Fig. 1, A and B ) revealed biphasic kinetics for cytochrome c-mediated electron transport at all ionic strengths except 0 mM ( Table I). The high affinity K,,, increased by an order of magnitude while the low affinity K,,, increased by 4 orders of magnitude as the ionic strength was raised from 0 to 150 mM revealing a decrease in the affinity of cytochrome c for its redox partners with increasing ionic strength ( Table I). These results for duroquinol oxidase activity in intact inner membranes are consistent with results obtained for purified beef heart ubiquinol-cytochrome c reductase (19) and cytochrome oxidase (20). The high affinity V,,, remained relatively constant from 25 to 100 mM ionic strength, whereas the low affinity VmaX increased with increasing ionic strength from 0 to 150 mM. Increases in the rate of electron transport mediated by cytochrome c occurred in both KCl/Hepes and KPi buffers indicating that the ionic strengthdependent rate of electron transport is not due to an anionspecific effect. These data reveal that with increasing ionic strength the rate of electron transport mediated by cytochrome c increases and the affinity of cytochrome c for its redox partners decreases while, as we have shown (15), the diffusion rate of cytochrome c increases.

Cytochrome c-mediated Electron Transport in Isolated Inner Membranes Compared to Whole Intact Mitochondria-
The duroquinol oxidase activities in both inner membranes and in intact mitochondria were measured over a broad range of ionic strengths (Fig. 2). Duroquinol oxidase activity of inner membranes, containing only endogenous cytochrome c, in-

~w t -
Reduction of endogenous cstochrome c in intact mitochondria at various ionic strengths. The enzymatic reduction of cytochrome c was measured at 550-540 nm wavelength pair during duroquinol reductase activity. The assay conditions were: 60 p~ duroquinol, 5 p~ rotenone, 1 p~ carbonyl cyanide m-chlorophenylhydrazone, 1 mM KCN. The total reduction of cytochrome c was measured by adding solid sodium dithionite. exogenous cytochrome c concentration of 100 PM, which is equal to its physiological concentration in the intact mitochondria, a greater enhancement and progressive increase in duroquinol oxidase activity occurred in inner membranes as ionic strength was increased from 0 to 150 mM. Duroquinol oxidase activity of intact mitochondria paralleled this progressive increase in the rate of electron transport with increasing ionic strength and showed, as with isolated inner membranes, that cytochrome c mediates maximum electron transport in the intact mitochondria at physiological ionic strength (Fig. 2). Thus duroquinol oxidase activity is greatest in both isolated inner membranes and intact mitochondria at physiological ionic strength where we have shown (15) using inner membranes that cytochrome c diffuses in three dimensions.

State Electron Transport in the Whole Intact Mitochondrion-
Considering the increase in the duroquinol oxidase activity with increasing ionic strength, in intact mitochondria, the question arises, does all the cytochrome c of an intact mitochondrion participate in steady state electron transport at low ionic strength, when it is membrane-bound and diffusing laterally and at high ionic strength, when it is not membranebound and is diffusing in three dimensions? To determine whether cytochrome c functions in a common pool for all of its diffusion modes, we measured the percent reduction of endogenous cytochrome c in intact mitochondria between 0 and 150 mM ionic strength, using either succinate or duroquinol as substrate, followed by the addition of KCN (Fig. 3). It was determined that more than 95% of the cytochrome c was enzymatically reducible with either substrate compared to its total reduction by sodium dithionite for all ionic strengths studied. These data indicate that all of the endogenous cytochrome c in intact mitochondria participate in steady state electron transport irrespective of its diffusion mode.

Characteristics of Electron Transport in Isolated Inner
Membranes Related to Cytochrome c Diffusion-Cytochrome c-mediated steady state duroquinol oxidase activity can be divided into four major distinct reaction (electron transfer) and diffusion steps: the reaction of oxidized cytochrome c with reduced cytochrome bc,; the diffusion of reduced cytochrome c to oxidized cytochrome oxidase; the reaction of reduced cytochrome c with oxidized cytochrome oxidase; and the diffusion of oxidized cytochrome c back to reduce cytochrome bcl. In these steps, the steady state reaction (electron transfer) rates of duroquinol reductase and cytochrome oxidase activity are 3-7 times faster than the overall duroquinol oxidase activity (19)(20)(21). The maximum reaction rate of the reduction of oxidized cytochrome c by reduced cytochrome bcl is 480-590 e-/s (19) and the maximum rate of oxidation of reduced cytochrome c by oxidized cytochrome oxidase is even higher (20, 21). From these reaction rate data, it is clear that the slowest, or rate-limiting step, in the overall duroquinol oxidase activity is the transport of electrons between cytochrome bc, and cytochrome oxidase by cytochrome c, i.e. the diffusion step of cytochrome c.
To more directly ascertain the relationship of cytochrome c diffusion to the overall kinetic process of electron transport, we determined the apparent K , values of cytochrome c and the maximum rates ( VmaJ of duroquinol oxidase activity in inner membranes at various ionic strengths. Our data showed a decrease in the affinity of cytochrome c for its redox partners and an increase in Vmax for duroquinol oxidase activity when the ionic strength of the medium was increased from 0 to 150 mM. These findings are consistent with a decrease in the electrostatic interaction of cytochrome c for its redox partners and consequently a shorter binding interval of cytochrome c for the active sites of its redox partners as ionic strength is increased as predicted by theoretical calculations (22,23). These data, combined with our diffusion and binding data (15), show that a decreased affinity and an increased rate of diffusion of cytochrome c, which occur as ionic strength is raised, results in an increased rate of electron transport.

Electron Transport and Cytochrome c Diffusion in Isolated Inner Membranes Compared to Whole Intact Mitochondriu-
We determined that the duroquinol oxidase activity of inner membranes containing only endogenous cytochrome c increased with increasing ionic strength up to 50 mM. This can be correlated with the faster pseudo-lateral diffusion of cytochrome c at 50 mM compared to lower ionic strengths for inner membranes (15). At higher ionic strengths, endogenous cytochrome c leaves the surface of the inner membrane, consistent with our fluorescence recovery after photobleaching, resonance energy transfer by Forster mechanism, and direct binding data (15), to become so diluted that it is unavailable for electron transport. It is logical that in the intact mitochondria, cytochrome c leaves the surface of the inner membrane above 50 mM ionic strength. However, the maximum volume available for three-dimensionally diffusing cytochrome c in the intact mitochondria is limited by the ionpermeable outer membrane, thus, its duroquinol oxidase activity does not decrease with increasing ionic strength but rather increases. Our kinetic data (Table I, Fig. 2) on the duroquinol oxidase activity in inner membranes (Table I) indicates that this increase in the rate of cytochrome cmediated electron transport with increasing ionic strength in intact mitochondria and in cytochrome c supplemented inner membranes as well, is due to three-dimensionally diffusing (low affinity) cytochrome c and not due to small quantities of membrane-bound (high affinity) cytochrome c. The increase in duroquinol oxidase activity with increasing ionic strength parallels an increase in the low affinity V,,, for electron transport and a large increase in the low affinity K, of cytochrome c. In contrast, the small increase in the high affinity K,,, of cytochrome c and relatively constant high affinity V, , of electron transport indicate that electron transport cannot be maintained by small quantities of high affinity membrane-bound cytochrome c.
The parallel maximal duroquinol oxidase activity which occurs at physiological ionic strength in both inner membranes and in intact mitochondria, requires a naturally high concentration of cytochrome c. This finding suggests to us that the decrease in the electrostatic interaction of cytochrome c with the inner membrane at physiological ionic strength, which results in the three-dimensional diffusion of cytochrome c, is compensated for by the naturally high concentration of cytochrome c found in the intact mitochondria.
Pool Function of Freely Diffusible Cytochrome c-We reported cytochrome c to be freely diffusible at the inner membrane surface at all ionic strengths and for all modes of diffusion (15). In the present paper, we have shown that >95% of the cytochrome c in intact mitochondria is enzymatically reducible at all ionic strengths consistent with studies using single ionic strengths (24-26). These diffusion and enzymatic data indicate that all cytochrome c is freely diffusible in a common pool and participates in electron transport in intact mitochondria whether it is bound to the inner membrane surface and diffusing laterally at low ionic strength, or not bound to the inner membrane and diffusing in three dimensions at higher ionic strengths, including physiological.
A diffusible pool of cytochrome c has been proposed previously from kinetic studies of reconstituted ubiquinol oxidase (6). The mobility of cytochrome c has also been argued from a number of studies showing that the electron transfer interaction domains of cytochrome bcl, cytochrome oxidase, and cytochrome bs, are essentially the same, i.e. all bind to the same domain on the cytochrome c molecule (27-30). From these studies, it neither appears necessary nor indeed possible, for cytochrome c to interact simultaneously as a tertiary complex with its two redox partners during its electron trans-

TABLE I1
Diffusion-based collision efficiency of cytochrome c-mediated electron transport fer. Our data are consistent with a common pool function in which diffusion-based collisions of cytochrome c with its membrane-restricted redox partners mediate electron transport.

Diffusion-based Collision Efficiency of Cytochrome c-mediated Electron Transport-We have used the treatment of
Astumian and Chock (31) to determine the theoretical diffusion-based collision frequencies in a heterogenous diffusion system between potentially active cytochrome c and its redox partners, cytochrome bc, and cytochrome oxidase, in collisions/s/cm2 of inner membrane (15). Using these collision frequencies (shown in Table 11, column 6) we can derive the collision frequencies of one active redox molecule with its active redox partner (column 7), which can be compared to its electron transfer frequencies or turnover numbers (column 8) to obtain its collisions/turnover (column 9) and collision efficiencies (column 10) for all three diffusional modes of cytochrome c.
This new diffusion and electron transport data and treatment thereof ( Table 11) show that the collision frequencies (column 7) of cytochrome c with cytochrome bc, and cytochrome oxidase are closer to their respective experimentally determined electron transport frequencies (column 8) for all diffusion rates (column 4) and all diffusion modes of cytochrome c (column 5). With increasing ionic strength, the diffusion rate of cytochrome c increases (column 4), the collision frequencies of all redox partners decrease (column 7), their electron transport frequencies increase (column 8), and their collision efficiencies increase (column 10). Concomitantly, the affinity (from Kd, resonance energy transfer, and binding data) of cytochrome c for the inner membrane decreases with increasing ionic strength (15). At physiological ionic strength, where the diffusion rate of cytochrome c is maximal and three-dimensional, and its affinity for the inner membrane is lowest, its diffusion-based collision efficiency with either of its redox partners is highest, approaching values consistent with diffusion control (column 10).
These results underscore the contributions of both diffusion and concentration (affinity) of cytochrome c near the surface of the inner membrane in the overall electron transport process mediated by cytochrome c. As indicated in our companion paper (15), at lower ionic strengths, where the diffusion rate of cytochrome c is lower, its collision frequency with its redox partners is higher since its concentration near the inner membrane and its redox partners is also higher. At lower ionic strength, the occupancy time of cytochrome c in the vicinity of the active site of the redox partner before and after the transfer of an electron is greater. Thus for steric reasons, this decreases the probability for a subsequent, effective collision, resulting in a lower rate of electron transport, therefore, a lower collision efficiency. In contrast, at higher and physiological ionic strengths, where the diffusion rate of cytochrome c is higher and three-dimensional, its collision frequency with its redox partners is lower since its concentration near the inner membrane and its redox partners is also lower. At physiological ionic strength, the occupancy time of cytochrome c in the vicinity of the active site of the redox partner before and after the transfer of an electron is shorter. Thus, at physiological ionic strength there is a high probability for a subsequent, effective collision resulting in a higher rate of electron transport, therefore, a higher collision efficiency.
These findings are consistent with a conformational basis for association and dissociation of redox partners in the random collision model of mitochondrial electron transport. Redox proteins appear to exist in various conformations (32, 33). Thus, for any diffusional mode of cytochrome c, a productive, i.e. electron transferring, collision is thought to require a conformation-specific association between the active site of cytochrome c and its redox partners. It follows that a concerted conformational change occurs in the redox partners concomitant with electron transfer which, we believe, is the basis for an electron-induced, conformation-driven dissociation from the active site of such redox partners. This dissociation is required before a subsequent, diffusion-based, productive collision can occur.
In summary, our findings reveal that for any of the three diffusion modes of cytochrome c, the diffusion-based collisions of cytochrome c with its redox partners have a direct influence on the overall kinetic process of electron transport. Furthermore, our findings reveal that at physiological ionic strength, cytochrome c diffuses rapidly in three dimensions and has a low affinity for the inner membrane to mediate the highest rate of electron transport through maximum collision efficiencies with its redox partners. The collision efficiencies reveal that the diffusion rate and concentration of cytochrome c near the membrane surface are rate-limiting for maximal (uncoupled) electron transport activity, approaching diffusion control.