Hi Krutika,

I see two factors that could give what you observe: no product formation.

The P450 in your system is not the problem since you measure its voltagrams.

First factor: as you write: the O2 concentration available in solution. Most P450 enzymes have a high affinity when reduced for dioxygen. So even low dioxygen concentration in solution should sustain a good actviity of a P450 enzyme. Caution, some P450 enzymes, mainly of bacterial origin, may present a low affinity for dioxygen.  You didn't mention the P450 you look at.

Second factor: the ability for electrons to go from the electrode to the oxidize heme iron ion into the P450 enzyme. Most P450 enzymes have a deeply buried active site with no visible access from the surface of the protein for the substrate. For an electron, this may be the cause of some difficulties for the electron to reach the heme iron since it has to travel through two different media: the solution and the protein "wall" (if I may say so). Therefore, an electrochemical mediator is required. Papers published some twenty years ago by Ron Estabrook's group at Nashville have shown that adding to the incubation mixture cobalt(II)sepulchrate trichloride at a 1 mM concentration is suffisient to see electromotive hydroxylation of lauric acid. WIthout sepulchrate, there is NO reaction, though a clear voltametric signal is observed (as you).

I thus strongly suggest that you add this electrochemical mediator to your assay and, maybe, you'll get hdyroxylated product!

Be aware, that a better reaction rate was obtained when using, instead of the widl-type P450, an artificial fusion of the P450 with the soluble domain of NADPH-P450 reductase. This is because the binding of the reductase to a P450 would induce some local conformational change that make the heme iron reducible.

Best regards and good luck for your experiments

Pr Philippe Urban

Dear Prof. Urban,

Thank you so much for your insights. We are working with a plant P450 which is membrane bound. We have reconstituted it in a biomimetic system and are doing electrochemical investigations. Describing further our set up:

When we start out without N2 gas bubbling (completely aerobic environment), the Cyclic voltamogram has a very huge cathodic peak. Then we bubble N2 gas for around 10 minutes and get extremely neat and reproducible CV. From this we have been able to calculate the redox potentials and surface coverage of molecules etc. Just after doing this CV, in the same partially anaerobic anaerobic environment we add the substrate and try to detect products. We do not detect any products.

Should we try to add substrates directly to the electochemical set up without nitrogen gas bubbling and apply a potential for triggering catalysis? I will also try adding the electochemical mediator and see if it works. 

I am also pretty unclear about generation of hydrogen peroxide by P450s. Does the formation of H2O2 by P450s mean indirectly that the P450 is active? I see some papers where they try to use an AMplex Red kit to confirm presence of H2O2 but I do not really understand what it means. 

Dear Krutika

Thanks for you feedback, I appreciate .

Concerning N2 ... well, maybe it could be interesting to look at what happens (or not) when you do not bubble N2 at all, yes. I'll do that, just in case. I am pretty sure that your incubation solution is under constant magnetic stirring, right?

But maybe there are other reasons for absence of hydroxylated product formation depsite clear ability of the P450 redox center to answer in voltametry.

Let me tell you two old stories (never published). I imagine that your plant P450 is not purified from the plants but produced by some recombinant systems, yeast probably, right?

Once upon a time, we faced a MAJOR problem with one the numerous P450 enzymes we have expressed in yeast. Human CYP2E1 was giving a clearcut reduced-CO differential spectrum but NO activity. We finnaly found out that it was expressed in a locked non-functional state. To unlock it, we had to grow the yeast cells that were expressing the recombinant CYP2E1 with propanol added to the culture medium. Propanol was going inside the cell and inside CYP2E1 at its actiev site, precluding the formation of the locked nonfunctional state. It worked fine. Maybe it is what happens to you? I recognize it is a rare case but ... who knows?

Once upon another time, we produced human CYP2A6 in yeast at high amounts (judged from the reduced-CO spectrum). When incubating it with its substrate and NADPH, no activity. This was the case again and again until we pre-incubated the recombinant CYP2A6 with recombinant human cytochrome b5. This mix was giving a huge CYP2A6-catalyzed hydroxylase activity. Maybe it is the case for your problem?

Now, concerning H2O2. Yes, H2O2 production in a P450 reaction does mean indeed that the P450 is functional, at least to reduce dioxygen! But it isbad news for  its function with the substrate. H2O2 is produced from uncoupling reactions in the catalytic cycle of a P450 .. uncoupling steps are fighting against THE coupling step which is hydroxylated product formation. In other words, the more  H2O2 is produced, the less hydroxylated product is produced. I have attached a very clear and recent review on this by Dr Andie Munro at Manchester.

We have also published papers in which we were precisely dosing the H2O2 produced during CYP3 turnovers (Biochemistry 1998 37: 11412-11424). To do so, we used the conversion of homovanillic acid in its highly fluorescent dimer by a stoechimetric amount of H2O2 in the presnece of horseradish peroxidase. Typically in 1ml of incubation mixture to quantify, we add 0.5 mg of homovanillic acid and 2 micrograms of horseradish peroxidase. Reaction: 5 min at RT. AN aliquot is separated onto HPLC with fluorescence detection: Lambda EXC = 315 nm, Lambda EMM = 425 nm. It worked well.

Good luck for your experiments,

Philippe

Dear Prof. Urban,

Many thanks for the paper and the much valuable feedback.  It has allowed me to think about these experiments in a more broad fashion. 

Yes this is  a full length P450 we produced heterologously in yeast. Interestingly, the CO spectrum of the microsomes was good, but after purification on a HisTag column, it gave a 420 peak only. We have seen this for all our plant CYPs, that they give a 420 spectrum post-purification. However, when we test these purified enzymes by our radio-label activity assays we find them fully functional. They also retain activities when reconstituted into synthetic carriers like vesicles/nanodiscs. 

I am wondering if immobilization of these proteoliposomes on gold electrodes renders them inactive. The liposomes could bind to the electrode either by physisorption or by Au-S chemistry (our proteins have several surface exposed cysteines). I think this is less likely. 

But I will definitely try the experiments that you suggested and also may be do the Apmlex Red assay to determine the extent of uncoupling. Perhaps that is a reason of my problem. I will also let you know if there is anything interesting :)

Best regards,

Krutika

Caution with amplex Red.

We have found that with insect beculovirus recombinant microsomes we get a response also with blanks. Thus you need to verify your experiment well with different type of blanks and see if you get linear response with concentration and time.