Hydrogen Bonding to P700:  Site-Directed Mutagenesis of Threonine A739 of Photosystem I in Chlamydomonas reinhardtii^,

Abstract

The primary electron donor P700 of photosystem I is a dimer comprised of chlorophyll a (P(B)) and chlorophyll a' (P(A)). P(A) is involved in a hydrogen bond network with several surrounding amino acid residues and a nearby water molecule. To investigate the influence of hydrogen bond interactions on the properties of P700, the threonine at position A739, which donates a putative hydrogen bond to the 13(1)-keto group of P(A), was replaced with valine, histidine, and tyrosine in Chlamydomonas reinhardtii using site-directed mutagenesis. Growth of the mutants was not impaired. (i) The (P700(+)* - P700) FTIR difference spectra of the mutants lack a negative band at 1634 cm(-1) observed in the wild-type spectrum and instead exhibit a new negative band between 1658 and 1672 cm(-1) depending on the mutation. This band can therefore be assigned to the 13(1)-keto group of P(A) which is upshifted to higher frequencies upon removal of the hydrogen bond. (ii) The main bleaching band in the Q(y)() region of the (P700(+)* - P700) and ((3)P700 - P700) absorption difference spectra is blue shifted for the mutants by approximately 6 nm compared to that of the wild type. A blue shift is also observed for the main bleaching in the Soret region. (iii) The (P700(+)* - P700) CD difference spectrum of the wild type reveals two bands at 694 nm (positive CD) and 680 nm (negative CD) of approximately equal area. For each mutant, these two components are blue-shifted to the same extent. The results strongly suggest that a blue shift of the Q(y) absorption band of P(A) is responsible for a blue shift of the exciton bands. (iv) Redox titrations yielded a decrease in the midpoint potential for the oxidation of P700 by 32 mV for the exchange of Thr against Val. (v) ENDOR spectroscopy shows that the hfc of the methyl protons at position 12 of the spin-carrying Chl P(B) is decreased due to the removal of the hydrogen bond to P(A). This indicates a redistribution of spin density in P700(+)* compared to that in the wild type. This gives evidence for an electronic coupling between the two halves of the dimer in the wild type and mutants.

Mutation of the Putative Hydrogen-Bond Donor to P700 of Photosystem I

“…The intensity of the ∼680-nm band was somewhat variable and usually larger in particles (data not shown); it may contain contributions from bleached antenna chlorophylls. These results are in good agreement with those obtained previously by Witt et al (16) in both spectral regions. However, we note that the additional bleaching band at ∼678 nm is smaller in our mutant than in any of theirs.…”

“…However, we note that the additional bleaching band at ∼678 nm is smaller in our mutant than in any of theirs. Because the relative size of this band seems to decrease from the T739Y to the T739V mutant (16), it may be related to the strength of hydrogen bonding to P A . This is consistent with the claim that the T739A mutant has the most severe effect (see below).…”

15 N photochemically induced dynamic nuclear polarization magic-angle spinning NMR analysis of the electron donor of photosystem II

“…3, spectrum C), showing a strong decay of the emissive compared with the enhanced absorptive signals, suggests that the emissive signals originate from the donor Chl. The emissive signal at 211.5 ppm is broadened by a shoulder at Ϸ215 ppm (Table 1), indicating involvement of a second donor Chl cofactor, which can be due to the epimerization at the C-13 2 and the different hydrogen-bonding at the 13 1 -keto group (21), causing also the split of the N-IV signal (250.3 and 254.9 ppm), whereas no effect is observed on the remote N-I position (186.2 ppm). The resonance of N-III is observed at 193.2 ppm.…”

Mutation Induced Modulation of Hydrogen Bonding to P700 Studied Using FTIR Difference Spectroscopy

“…Two mechanisms could account for the lowered frequency: (1) the 13 3 ester CdO is coupled to the bridging ester oxygen, which is H-bonded ( Figure 2C). We note, however, that the ester oxygen is likely to be a weak H-bond acceptor (11). (2) The lowered frequency of the 13 3 ester CdO of P A could also result from the fact that P A is a Chla′ and/or the fact that ring V of P A (unlike P B ) is considerably bent out of the plane of the macrocycle ( Figure 2C,F).…”

“…However, this is likely to be the case only if an H-bond to the 13 1 keto CdO of P A is still present in the TA(A739) mutant. Witt et al have also concluded that an H-bond is still present in all three of their TA739 mutants (11). The H-bond in the mutant is possibly mediated by an introduced water molecule.…”