Hello Razia,

First of all, this "coupled CII+CIV activity" should not be written like this, because it gives the impression that electrons can be transferred via CII directly to CIV, which is of course not possible. The electron pathway from succinate to Oxygen is catalyzed by 3 enzymes and involves quinone and cytochrome c as redox carriers as you have pointed out. Thus, if you didn't add the cyanide, what you were following was the so called "succinate oxidase" activity.

However, since you have two enzymes sharing cytochrome c (CIII and CIV), you cannot trust in the values from this assay because you have a competition, on the one hand, reducing Cyt c by CIII and, on the other, oxidazing Cyt c by CIV and the molecule you're following at 500 nm is the reduced Cyt c only. The latter explains why you're getting a negative slope, essentially you're following the CIV activity (which btw is the fastest enzyme of the respiratory chain). With this in mind you'll never be sure to what extent the rates you're obtaining are reliable and comparable with other samples, protein amounts, etc. The reduction of the Cyt c can be different, therefore not giving always the same effective concentration and the same activity values.

You have to add cyanide to the assay in order to follow the reduction of Cyt c mediated by complexes II and III. By contrast, if you're interested in the CIV activity, you can either follow the oxidation of reduced Cyt c (spectrophotometry) or follow the oxygen consumption (Oxygraph) in the presence of succinate (and cyt c, if the mitochondrial membranes are broken).

Hope this answer helps you! I'd be happy to help you if you have further questions.

Best,

Alfredo

Hello Alfredo,

Thank you for the answer.

I agree, it shouldnt be written as coupled II+IV activity. Thanks for correcting.

I have few questions.

If I look at the protocol for complex IV activity as per the reference I have provided in the question, we follow the decrease in absorbance due to oxidation of reduced Cyt C at the same wavelength, 550nm.

Procedure:

1. The assay is performed at 550 nm following the decrease in absorbance resulting from the oxidation of reduced cytochrome c.

2. In 1 ml of isosmotic medium containing 10 mM KH2PO4, pH 6.5, 0.25 M sucrose, and 1 mg/ml BSA, add mitochondria (20–40μg of protein) or whole cells (3–10×104 cells) and 10μM reduced cytochrome c.

3. Follow the reaction for 3 min (rate 1).

4. Permeabilize the external mitochondrial membrane by adding 2.5 mM lauryl maltoside (rate 2).

5. Follow the reaction for 3 min. Inhibit the reaction with 240μM KCN.

So, I was wondering why the decrease in absorbance in the assay I did cant be considered as due to oxidized cyto C.

Isn't the reduction and subsequent oxidation of cytoC being carried out by ComIII and ComplexIV respectively, in a sequence rather than in a competing way.

When I analyzed the data with all the samples,(I finished the experiment and then realized what I missed in the buffer working stock), the difference between the groups is significant and it is complying with the respirometry data of complex IV analyzed by TMPD+Ascorbate using Agilent Seahorse. So I am curious to know if it is reliable and my data makes any sense at all.(Also curious to see if can save my experiment)

By the way, I added cyanide and did CII+III assay yesterday. As for complex IV activity, I waited too long for sodium dithionite to arrive from the vendor, so I had to cancel the order. Is there any other easy way to reduce cytochrome C. I read ascorbic acid and DTT can do it but couldnt get the detailed protocol.

I appreciate your willingness to help.

Cheers,

Razia

Dear Razia,

You're right, the decrease in absorbance in the assay you did corresponds majorly to the oxidation of Cyt c catalized by CIV. In your assay the source of electrons is Succinate, which feed electrons via CII to the quinone. The latter becomes reduced and is now a substrate for CIII, which transfer electrons to the cytochrome c (oxidized). Finally, resulting reduced Cyt c is used by CIV to reduce Oxygen into water. Thus, in a way you're right the redox reactions are sequential.

However, the exogenous cytochrome c (oxidized) you're adding to the assay is at the same time being reduced by CIII and oxidized by CIV, both processes are competing for this substrate. The only redox state of Cyt c absorbing at 550 nm is the reduced form. Since you're following this molecule only, you can't be sure that the resulting absorbance changes (rates) result exclusively from oxidation; at the same time CIII reduces a fraction of Cyt c (which absorbs at 550 nm) and those electrons are immediately transferred to CIV. Besides, CIV activity is usually higher than the one from CIII, therefore giving a short time of Cyt c to stay in its reduced form. You will never be sure that the reduced fraction of Cyt c is the same in all your experiments and conditions, particularly if you have variations of the respiratory complexes, protein amount, etc.

I'd suggest to repeat your experiment, because if you wanted to measure CII+CIII, basically the activity of CIV was not important for you. CII+CIII is helpful to determine differences in the activity of CII, and, under certain situations CIII. In this case CII is the limiting step of the redox transfer, thus it controls the flux.

If what you wanted to measure is the individual CIV activity, but you can't generate reduced Cyt c and don't have an Oxygraph machine, you can isolate mitochondria and run a BN-PAGE (or hrCN-PAGE) and stain it with Cyt c (oxidized) and diaminobenzidine (DAB); it results in a brown/black colored band. In this case, the electrons are transferred from DAB to cyt c where the CIV band is. It's quite specific and gives you an idea on the CIV content, which can be related to the activity of this complex.

I can help you further if you can give me more details. Please send me an inbox.

Best wishes,

Alfredo