I have a great respect for Dr. Yu as an experimentalist and I actually think a concerted reaction mechanism is quite likely, but I absolutely do not think this experiment proves it.
We already know the semiquinone at Qo cannot be detected by epr spectroscopy under oxidant-induced reduction conditions, much less initial steady state conditions. Suppose the sensitivity limit is 1% occupancy, this means the semiquinone must exist for less than 1% of the turnover cycle. Active bovine bc1 complex turns over at rates aproaching 1000 s-1. That means the lifetime of the semiquinone must be 10 usec or less, and after that time we expect both ISP and bL to be reduced. There is no point in doing this experiment unless you can resolve times shorter than that. And even then it wouldn't work unless you had a way to start all the complexes synchronously with a "jitter" in the start time less than the life of the semiquinone. Rapid mixing won't do it. Flash activation through reaction center won't do it due to the jitter in time for diffusion of c2 between reaction center and bc1 comlex. Ruthenium flash activation at c1 might have a chance, but due to the 10's of usec required for the Rieske to carry the electron hole from c1 to the Qo site, a negative result would be meaningless.
Dr. Yu makes the following analogy to people who question the time resolution of his technique: "You tell me some one is sitting in that chair. I tell you I can't see him. You tell me my eyes aren't good enough. I prefer to believe my eyes and I tell you no one is there."
That's fine when you have every reason to expect your technique is adequate to make the observation- as for eyes seeing nearby persons. But in this case the indetectability of the semiquinone already indicates the reaction is concerted to within something like 10 usec, so this elegant new experimental technique with time resolution ~100 usec would not be expected to separate reduction of ISP and cyt b, and indeed it didn't.
Another point- rapid freezing may freeze out large scale motions but not tunneling- Suppose the sample freezes at 250 usec with ISP reduced, both b's oxidized, and semiquinone in Qo ready to reduce cyt b (i.e. already moved to the proximal area of the pocket if that is in fact required). What is to keep the electron from hopping from the semiquinone to b(L) in the hours before you put the tube in the epr cavity? Does low temp freeze out that reaction? Or transfer from b(L) to b(H)?
Actually Figure 4a seems to show c1 and maybe b(H) going reduced a lot faster than b(L). This doesn't seem to be discussed, and it's not obvious where the electrons are coming from, unless by slow tunneling from ISC and B(L) during the intervening hours. (For assignment of the peaks in Fig 4a see Salerno, JBC 259,2331)
2 comments:
I have a great respect for Dr. Yu as an experimentalist and I actually think a concerted reaction mechanism is quite likely, but I absolutely do not think this experiment proves it.
We already know the semiquinone at Qo cannot be detected by epr spectroscopy under oxidant-induced reduction conditions, much less initial steady state conditions. Suppose the sensitivity limit is 1% occupancy, this means the semiquinone must exist for less than 1% of the turnover cycle. Active bovine bc1 complex turns over at rates aproaching 1000 s-1. That means the lifetime of the semiquinone must be 10 usec or less, and after that time we expect both ISP and bL to be reduced. There is no point in doing this experiment unless you can resolve times shorter than that. And even then it wouldn't work unless you had a way to start all the complexes synchronously with a "jitter" in the start time less than the life of the semiquinone. Rapid mixing won't do it. Flash activation through reaction center won't do it due to the jitter in time for diffusion of c2 between reaction center and bc1 comlex. Ruthenium flash activation at c1 might have a chance, but due to the 10's of usec required for the Rieske to carry the electron hole from c1 to the Qo site, a negative result would be meaningless.
Dr. Yu makes the following analogy to people who question the time resolution of his technique:
"You tell me some one is sitting in that chair. I tell you I can't see him. You tell me my eyes aren't good enough. I prefer to believe my eyes and I tell you no one is there."
That's fine when you have every reason to expect your technique is adequate to make the observation- as for eyes seeing nearby persons. But in this case the indetectability of the semiquinone already indicates the reaction is concerted to within something like 10 usec, so this elegant new experimental technique with time resolution ~100 usec would not be expected to separate reduction of ISP and cyt b, and indeed it didn't.
Ed B.
Another point- rapid freezing may freeze out large scale motions but not tunneling- Suppose the sample freezes at 250 usec with ISP reduced, both b's oxidized, and semiquinone in Qo ready to reduce cyt b (i.e. already moved to the proximal area of the pocket if that is in fact required). What is to keep the electron from hopping from the semiquinone to b(L) in the hours before you put the tube in the epr cavity? Does low temp freeze out that reaction? Or transfer from b(L) to b(H)?
Actually Figure 4a seems to show c1 and maybe b(H) going reduced a lot faster than b(L). This doesn't seem to be discussed, and it's not obvious where the electrons are coming from, unless by slow tunneling from ISC and B(L) during the intervening hours. (For assignment of the peaks in Fig 4a see Salerno, JBC 259,2331)
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