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title: Mechanism of the Pfr-photoreaction

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# Mechanism of the Pfr-photoreaction
## Observations in the wt
### Visible pump-probe spectroscopy:
* Majority of the excited population is quenched within 200 fs. Simultaneously, a red-shifted ground-state like product appears. 
* Red-shifted product has a strong coherent modulation which we can assign to low-frequency modes involving the Ring-D and the CD-methine-bridge (in H2O around 300 cm-1 or about 111 fs).
* Within one and four ps, the small remainder of the excited state population also decays, likely also refilling the red-shifted product and ground state.
* Red-shifted product decays with ~4 ps to ground state. Shape of the constant component appears to be pH dependent. Either red-shifted or blue-shifted (lower pH)

His-278 mutants affect the photo-reaction strongly. The dominant 200 fs dynamics is missing. For H278Q at low pH, mostly a long lived excited-state is observed (hunderts of ps). With increasing pH, wt like behavior seems to be recovered. 


### IR pump-probe spectroscopy:
* Continuum band rising with less than 300 fs (observable up to >1800 cm-1). Decays with ps (mostly 1 ps).
* Bleaching dominated C=C region. Some bands indicate very fast dynamics (but difficult to quantify due to time-zero in the IR).  Shows dynamics with 1 ps. Decays with 4ps.  
* Final Ring-D product likely appears with 1 ps.
* Prop-c band appears almost immediately. Quite broad, not changing at 1 ps scale and ESA area approximately equals BL area, hence unlikely deprotonated.
## Possible Mechanisms
### 1. Deprotonation of chromophore
* Explains ultrafast quenching
* Explains CB band by assuming the water cluster between the propionate side-chains is the the proton-acceptor. Alternatives are other waters near the chromophore. Pyrole $H_3O$?
* Could explain Bleach-dominated C=C spectra. Comparison of the calculated protonated and deprotonated IR-spectra should be informative
* Explains pH and mutation influence. Either directly affects part of the transport chain or indirectly by shifting the pKa of one amino-acid in the chain. H278 and Y165 mutants disrupt the water network between propB and propC, the assumed proton acceptor group. Since ultrafast proton transfer depends critically on distances, an increase of distance could diminish the reaction probability?
* Red-shifted product: Can it be explained by chromophore deprotonation? **Is it possible to calculate the S$_0$→S$_1$ transition for the deprotonated chromopore? Can we compare the pKa of the pyrrole NH groups in the excited-state and the ground-state?**
* pKa values of histidines and other AA?
* Origin of the proton: likely ring-C. Shuttled away by the pyrrole water and His-248? Other routes are also possible. Or is His-278 involved? Distance to potential donors is quite large. Distance is ? 
* If the CB is caused by the waters between the proptionates, why does it not react strongly to the CB decay on the 1 ps scale? The prop-C C=O behavior can be explained by the deprotonated chromophore alone according to Marias calculations.
* Can we rule out ring-D? Maybe deprotonation via Asp makes rotation of the ring possible?
-> Unlikely, that Asp in vicinity of ring D accepts the proton and is responsible with water molecules for the CB: because ring D forms its isomerized product within 1 ps (or faster), and the CB decays with 1 ps. The rotated ring D is too far away from the asp group to re-accept the proton in the Lumi-F.


##### Effect of Ring-C deprotonation on calculated IR spectra
Based on Marias and Duc calculations, which were done in $H_2O$


#### Proposal from Peter

1. Upon electronic excitation, a proton is released from the BV to a water cluster (within 300 fs). We do not know a priori where the proton comes from but we can now rule out the propC. Thus, it is very likely that it originates from an N-H group of the BV.
2. In the ground state, the most acidic N-H group is at ring B/C, but in the electronically excited state it can be different, depending on the charge distribution in the excited state. I think it is more likely that the pKa of the ring D N-H group is affected in the excited state rather those of ring B or C (Igor may comment on this). Moreover, intuitively I would expect that such a chromophore with a positive charge at ring B/C and negative charge at ring D may give a red shifted ESA.

_Comment to the ESA: Upon excitation to $S_1$ the electron density is shifted towards ring B. As a consequence the double bond character at the C-D methine bridge is reduced (SI Figure S1 b) facilitating the isomerization of ring D. In my view this could disagree with a deprotonation of ring D. In Figure S1b we plotted the electron density differences. 
**IGOR**: Is it possibel to calculate the atom distances in the $S_1$ compared to $S_0$? This would help to argue on strengthening and weakening of bonds..._

4. Assuming that a proton is released from the ring D nitrogen, it can readily be channeled to the water network in the vicinity of His278, Y165, and propC. Short distances for proton transfer.
5. This interpretation can account for effect of the His278 mutations. Substituting His by Q (or A) does not lead to the proton transfer at neutral pH, indicating that His278 may be involved in the proton transfer from ringD-N H to the water cluster. Only if we increase the pH sufficiently high, also the Q278 variant is capable to do the same job as His278 in the WT - not surprising since at sufficiently high pKa the proton may go directly to the water or transiently to the NH group of Q.
6. Now comes the strange behaviour of propC. 
    (a) the entire process of proton translocation described above may be assisted by propC which   undergoes a reorientation upon electron excitation in the excited state such that H-bonding interactions are different (frequency shift). 
    (b) the reorientation of propC is the consequence of proton transfer to the surrounding water network. Actually, the lifetimes favor option (a) and I like it more since can also account  for the RR activity of the C=O stretching mode.
    ( c ) another explanation could be based on Ducs calculations: if deprotonation of the chromophore in the ES changes the bond length or force constant of propC's C=O, this would be visible in RR spectra, too... Peter is this correct?



### 2. Deprotonation of a side-chain or AA via electric field effect or ultrafast structural changes
* Does not directly explain the ultrafast quenching and the influence of the mutations.
* Connection to the red-shifted feature?
* IR spectrum show no clear AA contributions from a possible donor
* 

### 3. Coupled charge transfer and isomerization (twisted CT)
* Subtype of 1.

### 4. Ultrafast hot product generation by ultrafast isomerization via CI
* Explains the red-product: very hot chromophore
* Does not explain CB in the IR. 
* Why is the reaction so sensitive to the mutation of His?

## Generalization to other Phytochromes

Similarities and differences to other phytochromes. Is this unique to AGP2 or does this mechanism to generalize to other phytochromes as well? 
a) Red-feature in the visible is at least also observed in Cph1-Pfr. The fast time-constant seems to be missing in Cph1 (has to be checked); ESA features seem to be different in dynamics.
b) At a first glance, time-resolved IR spectra in Agp2 are comparable to Agp1 (Diller); we could also measure Agp1 in D2O in the IR to see if a CB appears there too. We have to analyze our VIS data on Agp1.

