A simplified version of the redox chain of beef heart SMPs showing only the major electron transporting cofactors is illustrated in Fig. 1. About 50% of bovine heart SMPs in a given preparation are inside-out , and in this population the NADH:ubiquinol oxido-reductase (complex I) should be directly assessable to the electron donor NADH. The other electron-donating substance used in this experiment is succinate. Succinate is transported across the SMP membrane and should thus be capable of donating electrons directly to complex II (succinate:ubiquinol oxidoreductase) in both right-side and inside-out vesicles.
At this point, it is useful to mention the sites where inhibition of respiration can occur (see Fig. 1). Rotenone binds to the ubiquinone (Q)-docking site at complex I (*1), preventing electron flow from NADH via the iron-sulfur clusters (FeS) to the Q pool . Antimycin A binds near cytochrome bH (*2) in complex III and blocks the forward oxidation of reduced cytochrome bH and promotes reverse electron transfer from reduced bH to semi-ubiquinone . Cyanide binds at oxidized cytochrome a3 in complex IV (*3) and stops electron flow to the terminal electron acceptor, oxygen . Malonate is a compound that structurally resembles succinate and is therefore a competitive inhibitor of complex II (*4) .
Fig. 2 shows oxidized (blue) absorbance spectra of 0.3 mg/ml cyt c. The red trace shows cyt c after reduction with ascorbate. The major difference between the two spectra occurs in the region of the α-band at 550 nm. The micromolar extinction coefficient for reduced horse heart cyt c in 0.1 M phosphate buffer, pH 6.8, is 23.94 mM−1 cm−1 at 550 nm .
Table I denotes how the series of assays to be performed by students in this part of the experiment are to be set up. The reagents listed through rotenone in Table I may be pipetted into test tubes ahead of time (the electron-donating substances are not added yet). SMPs are added (one test tube at a time) at time (T) = 0:00 min. The absorbance is then recorded at 550 nm every 30 s for a 3-min time period to determine the endogenous rate of cyt c reduction. At T = 2:45 min, the electron-donating substance (either succinate or NADH) is added and the absorbance measured for another 3 min. Actual data obtained by a single undergraduate student (A. Melin) is recorded in Table II.
Fig. 3 shows the results obtained when SMPs were incubated with succinate (Table II; test tubes 1–3). In the absence of cyanide (□), little build up of reduced cyt c is observed, because electrons can immediately flow from reduced cyt c through complex IV due to the high affinity of cyt c for the respiratory complexes. However, when the flow of electrons from cyt c to complex IV is blocked with cyanide, the level of the cyt c population steadily increases and is observed as an increase in absorbance at 500 nm (▪). The slope of the initial linear portion of this reduction curve (initial rate) is used to estimate the rate of ET. It is interesting to note that when half the concentration of SMPs is added to the assay mixture (▴), the initial rate of cyt c reduction decreases by 50%. This illustrates to students the catalytic nature of the respiratory chain (the rate increases proportionally to the amount of SMPs).
Fig. 4 shows the results obtained in assays 4–6 of Table II (succinate + CN in the presents of inhibitors) with assay 2 included for comparison (succinate + CN only). Here it is possible to observe a significant inhibition of electron flow from succinate to cyt c in the presence of the competitive inhibitor malonate (a structural analogue of succinate). The respiratory inhibitor antimycin A strongly inhibits ET in SMPs. An unexpected result, however, was the observation that the complex I inhibitor rotenone also could inhibit electron flow between succinate and cyt c. To explain this possibility to students, it is necessary to inform them in the theory section of the laboratory manual of a previous reference , which showed antimycin A is capable of binding at the rotenone inhibition site in complex I. These results simply suggest that the reverse may be also be possible (rotenone can bind the antimycin A site in complex III).
Fig. 5 (Table II; test tubes 7–12) compares the rate of cyt c reduction when NADH is used as an electron donor. In the absence of CN (□), this rate is negligible because electrons from reduced cyt c are quickly transferred to complex IV (as was also observed in Fig. 3 with succinate). In the presence of CN (▪), we get a rapid rate of cyt c reduction, again confirming results observed in Fig. 3. Malonate, the competitive inhibitor of complex II, has no effect on the rate of cyt c reduction because complex II is no longer part of the electron flow pathway (▵). Unexpected results, however, are observed in Fig. 5 with the respiratory inhibitors rotenone and antimycin A (assayed separately) (○, •). Neither inhibitor had any effect on electron flow between NADH and cyt c. Our results suggest that electrons from NADH enter complex I and are then capable of reducing cyt c directly instead of indirectly through the ubiquinone pool and complex III. Again, students should be informed in the theory section of their laboratory manual of a report in the literature  using bovine heart SMPs that confirm our observation that complex I does display cyt c reductase activity. This activity can be inhibited by the compound adriamycin, also known as the cancer drug Doxorubicin . Unfortunately, Doxorubicin is extremely expensive from commercial sources (Sigma quotes $209 U.S./10 mg) and was not tested in this study. It may be possible to purchase Doxorubicin from hospital pharmacies at a cheaper rate. Biochemistry instructors are also welcome to omit the NADH assays if they feel the material adds too many complications to the experiment.
When writing up their reports, students will be asked to first subtract the endogenous rates from the initial velocity values they observe when succinate or NADH is added (slope of the initial linear portion of the cyt c reduction curve). From these initial velocities, it is possible to calculate the percentage rates observed in the presence of inhibitors relative to the succinate + CN rate, which is set to a maximum of 100%. Students can also calculate the reduction rates as micromoles of cyt c reduced per minute per milliliter using Beer's Law:
where A is the absorbance value at 550 nm, E is the extinction coefficient for reduced cyt c (reported to be 23.94 mM−1 cm−1 ), b is the path length of the curvette (1 cm), and c is the unknown concentration. For example, if the absorbance rate A = 0.046 units/min, c = A/Eb, or 0.046/23.94 mM−1 cm−1 × 1 cm. The value obtained should be divided by 1000 to convert to a molar concentration as the extinction coefficient is mM. From this we get a cyt c reduction rate of 1.92 μmol/min.
In this inquiry-based portion of the laboratory, students will try to construct a soluble redox chain between NADH (−0.32 V) and cyt c (+0.25 V). Because NADH is a two-electron donor/acceptor and cyt c is only capable of donating or accepting one electron at a time, students will need to try to use redox dyes as electron shuttles to accomplish the task. Students will test the dyes FCN, DCPIP, PMS, and TMPD. The structures of these dyes (FCN (ntp server: niehs.nih.gov), TMPD (11), PMS (11), DCPIP (1)) and their redox potentials (2) can be seen in Fig. 6. Some dyes are capable of accepting and donating only one electron at a time (n = 1). These redox chemicals will not function as an intermediary link between NADH and cyt c (FCN is an example). Other dyes may be capable of accepting or donating either one or two electrons at a time (n = 2), but their redox potential may be below that of cyt c (TMPD is an example).
It is up to the students to design the assays (including controls) for this section of the laboratory. A sample of the pre-laboratory assignment is shown in Table III. In the control assay, the absorbance of cyt c should be monitored for several minutes to establish an endogenous rate, and NADH should be added in order to see if electron transfer (even a slow rate) is possible. In separate test tubes (assays 2–5), students should test, one at a time, whether or not the redox dyes can reduce cyt c. Then, at T = 3:00 min, NADH is added and the rate monitored again for 3 min to see if the dyes can shuttle electrons between NADH and cyt c. The concentration of the redox dyes used in this experiment is 0.4 mM, which is one-tenth that of NADH. The most successful dye should be studied in ôone-tenth the concentration (0.04 mM). Students can do this dilution themselves. Experimental results that were obtained in this part of the experiment are recorded in Table IV and are illustrated in Fig. 7. Here it can be seen that a slow reduction of cyt c is possible with DCPIP but that PMS gives even better results, even when diluted 10-fold (0.04 M concentration).
At the end of this study, students should compare the structure of PMS with the structures of flavins or ubiquinone (found in the coenzyme section of all biochemistry text books) in order to understand how this redox dye might function as an electon shuttle between NADH and cyt c. In the end, they should draw the soluble redox chain constructed in a similar manner as is illustrated in Fig. 8.