Estados Unidos

DNA analysis of the major genes of PSI has not been successful in identifying the suppressor mutation of D576L. EPR analysis of the original mutant suggests that PsaC and its binding properties are effected in some way. It was expected that in the suppressor mutants the stability of PsaC would have changed, due to changes in PsaA, PsaB or PsaC. Sequencing of the genes encoding these proteins has shown that they are unchanged from wild type, and are not responsible the changed photosynthetic competence of the suppressor mutants. While it is possible that changes may have occurred in other parts of psaA or psaB, which were only partially sequenced, it is very time consuming completely sequenced two such large genes. It would be beneficial to be able to identify the region of PSI that is effected in the D576L mutant aud in the suppressor mutants. This could then provide information about which subunits (or part of subunits) are involved. Examination of electron flow through the suppressor PSI complexes was examined so that the effected areas could be identified.

- "■ -I---1---?--- r — ---1

10 2 0 3 0 4 0 5 0

W t T A C T A A A T T A T G C A T T CTTT GAACT A< 3 TTTTTTAr TTT AT< 3 T€ GTTACTT

Supl T A CT AA AT T A TG C A TT C NT T G A A C T A N T T T T T T A T T T N A T G T C G T T A C N T

Sup2 T A C T A A A T T A T G C A T T C T T T G A AC T A GT TT TT T A T T TT AT G T C GT T A C TT

Sup3 TACTAAATTATGCATTCTTT GAACT AGNNNNNT ANNNNNT GTCGTTACTT

Sup4 T A C T A A A T TA T G C A T T CT T T GA AC T AG T T TT TT A T T T T AT GT C G T TA C T T ' '■ "" T ' 4 ' "" "" ' ’‘'""-I "... "" 1‘ ' t 6 0 7 0 80 9 0 1 00 Wt TAAGAATTAA G A A T T A C T T T A T G A A A G A T T T T A C T A C T T A T T T A T C A A C A i Supl TAAGAATTAAGAA T T A C T T T A T GA A A GA T T TT A C T A C T T A T T T A T C A A C A Sup2 TAAGAATTAA G A A T T A C T T T A T G A A A G A T T T T A C T A C T T A T T T A T C A A C A Sup3 TAAOAATTA A GA A T T A C T T T A T GA A A GA T T T T A C T A C T T A T T T A T C A A C A Sup4 tA A G A A T T A A G A A T T A C T T T A T G A A A G A T T T T A C T A C T T A T T T A T C A A C A 11 0 1 2 0 ï i ô 1 4 0 150! wt GCACCTGTAATTGCTACTATTTGGT TTACTTTTACAGCAGGTTTATTAAT

Supl GCACCTGTAATTGCTACTATTTGGT TTACTTTTACAGCAGGTTTATTAAT

Sup2 G C A C C T GTAATTGCTACTATTTGGT TTACTTTTACAGCAGGTTTATTAAT

Sup3 G C A CCTGTAATTGCTACTATTTGGT TTACTTTTACAGCAGGTTTATTAAT

Sup4 GCACCTGTAATTGCTACTATTTGGT TTACTTTTACAGCAGGTTTATTAAT

Ï S0 1 7 0 180 1 30 200 wt T G A A AT T A A C C G T T A T T T C C C T G A T C C A C T T G T A T T T T C A T T T T A A T T A A Supl T G A AA TT A A C C G T T A T T T C C C T G A T C C A C T TG T A T T T T C A T T T T A A T T A A Sup2 T G AA AT T A A C C O T T A T T T C C C T G A T C C A C T T G T A T T T T C A T T T T A A T T A A Sup3 T G AA AT T A A C C G T T A T T T C C C T G A T C C A C T T G T A T T T T C A T T T T A A T T A A Sup4 T G AA AT TA A C C G T T A T T T C C C T G A T C C A C T T G T A T T T T C A T T T T A A T T A A 210 2 2 0 23 0 2 4 0 25 0 AATTGTGGATAACCTGATAATTATGACTGACACACTGGAGTGTGTCAGTC AATTGTGGATAACCTGATAATTATGACTGACACACTGGAGTGTGTCAGTC AATTGTGGATAACCTGATAATTATGACTGACACACTGGAGTGTGTCAGTC AATTGTGGATAACCTGATAATTATGACTGACACACTGGAGTGTGTCAGTC 2 60 zJd 2 8 0 290 wt ACATTTATTTACCCTCACGGTGGAAATAAGCTAT TTCAAACTATACT

Supl ACATTTATTTACCCTCACGGTGGAAATAAGCTAT TTCAAACTATACT

Sup2 ACAT T T ATTTACCCTCACGGTGGAAATAAGCTAT TTCAAACTATACT

Sup3 ACAT T T AT T T ACCCTCACGGTGGAAATAAGCTAT TTCAAACTATACT

Sup4 ACAT T T AT T T ACCCTCACGGTGGAAATAAGCTAT TTCAAACTATACT

Figure 3 .1 3 , The p s ^ sequences obtained from the suppressor mutants are shown WT- wild type, Sup 1-4- suppressor mutants 1-4,

3.5.1 F^/Fg reduction in the Suppressor Mutants

EPR spectra of Fa/Fb reduction was performed on the thylakoid membranes of all four suppressor mutants. The spectra obtained are shown in figure 3.14. It was expected that the spectra of the suppressor mutants would have reverted back to wild type values, but this clearly not the case. EPR spectra for the reduction of Fa/Fb in the suppressor mutants are essentially the same as the equivalent EPR spectra of the D576L mutant. They have not reverted back to wild type spectra. However, the suppressor mutants are able to grow photosynthetically (although at lower rates than wild type). A change in the binding of PsaC, as was originally assumed, cannot be the reason for the reinstatement of photosynthetic ability in the suppressor mutants.

3.5.2 Oxidation of Aj in the Suppressor Mutants.

Forward electron transfer kinetics in PSI are difficult to measure because of the high chlorophyll concentration associated with the reaction centre, and the small extinction coefficients of the quinone and the iron-sulphur centres. The oxidation of A,, however, can be determined, either by forward electron transfer to Fx or by back reaction with P700^. Forward electron transfer can only be measured at temperatures above 23OK, while the back reaction is seen at all temperatures in the appropriate sample redox state. The rate of the forward reaction at room temperature has been found to be in the range of 2-300ns in spinach by EPR and optical spectroscopic techniques at 293K. The back reaction rate varies in different preparations, and with the redox state of the iron-sulphur acceptors, and is measured to be around 10 ps and 100-150 ps.

Pulsed EPR was used to examine the oxidation of A,. Using this method, the spin polarised P700/A," radical pair is detected and its decay measured. However, at times longer than a few microseconds, it is not possible to distinguish between changes in the concentration o f the radicals and the decay of polarisation. Therefore, detection of the slower rate was not attempted.

D576L sup4

D576L sup3

'vYk D576L sup2

D576L supl

D576L

u\\fVwV4vM Wild Type

332.0

301.9 361.9

Field mT

Figure 3.14. Wide scan EPR spectra in the g=2.00 region of C. reinhardtii unffactionated thylakoid membrane fragments. Samples were prepared and EPR spectra recorded as described in figure 3.3. The spectra shown are light minus dark difference spectra. Chlorophyll concentration

The rate of forward electron transfer in wild type C. reinhardtii chloroplast membranes was measured. Rates for C. reinhardtii spinach membranes are similar (see table 3.1). The rate of A f reoxidation in the D576L mutant is less than wild type, but greater than other mutants lacking PSI. In the suppressor mutants, rates of A,' reoxidation are similar to that for wild type, in the range of 200-3 00ns.

3.5.3 Recombination Rates between P700^ and A { in the Suppressor Mutants. Electron transport in PSI is effected by temperature in a peculiar way. In the experiments described above, the temperatures were at room temperature and just below freezing. At these temperatures, forward electron transfer to the bound iron-sulphur centres is rapid and back reactions from F^/Fg to P700^ occur in milliseconds. As temperature decreases, the back reactions are inhibited, and P700 photooxidation appears irreversible. As temperature falls below 200K, the efficiency of forward electron transfer also declines. If illumination is continuous, irreversible oxidation of P700 and the reduction of F^/Fg is detected, even at 4K. If, however, a single turnover flash is provided, few reaction centres possess this stable charge separated state. In the majority of the reaction centres, a recombination between P700^ and A,' is seen. The recombination rate at lOOK in wild type and the suppressor mutants was measured. The results of this can be found in table 3.2. The recombination rate in wild type C. reinhardtii is faster than that of spinach, being 14-18 ps compared with 25-30 ps in spinach. This rate is unchanged in the D576L mutant, and in the suppressor mutants.