Sub-Introduction H

Use of Ti Saturation Recoveiy Measurements to Study the Stmcture of

Photosystem I

At the present time there is no complete high resolution X - ray crystal structure of photosystem I or any of its bacterial analogues, such as green sulphur bacterial reaction centres. The PSI of Synechococcus sp. has been crystallised but to date the structure has only been solved to a resolution of about 6 Â, which is too low for the reliable identification of chlorophyll much less of quinone molecules (Krauss et al, 1993). However the structure of part of the acceptor side can be accurately solved and this is because the three iron - sulphur clusters located there represent electron - dense regions which diffract X - rays well. This portion of the structure is shown schematically in figure 3.1, giving the distances in angstroms between the iron - sulphur clusters. The rest of the structure cannot be accepted with such confidence at this resolution and there is scope for the application of techniques other than X - ray crystallography to estimate the distances separating the other acceptors.

Hecks et al, 1994 have used picosecond photovoltage measurements to make deductions about the positions of the Aq and A^ acceptors relative to P700. Electroluminescence kinetics studies have also been used to obtain structural information about the PSI reaction centre (Vos and van Gorkom,|l988). In the following section a study

using Ti saturation recovery measurements to gain structural insights into higher plant PSI is presented.

3.1.1 The Principles of Saturation Recoveiy

The saturation of paramagnetic spin systems by microwave irradiation has been widely used to study electronic and structural aspects of such systems. In a saturation recovery experiment a spin system is irradiated with a short-lived high intensity microwave pulse, the kinetics of the recovery process by which the magnetisation reestablishes its thermal equilibrium are followed. Equilibrium is restored by relaxation, which can be defined as an energy exchange occurring either between spin systems (as in spin-spin coupling) or between a spin system and the surrounding crystal lattice (as in spin-lattice relaxation). Measuring the kinetics of the former yields a value for the parameter T2; measuring the kinetics of the latter

yields a value for T j. There are a number of unquantifiable factors that influence T2 relaxation

kinetics and for this reason T, is a purer parameter.

The radical is exposed to a pulse of high intensity microwaves for long enough to saturate the bulk magnetisation of the radical. The bulk magnetisation consists of the net contribution from electron spins, including those spins which are aligned against the applied magnetic field. Probing microwave pulses of lower intensity are used to phase the relaxing magnetisation. When the system is restored to equilibrium the input microwaves are released in a burst which results in a spin echo. The increase in the intensity of this echo is measured against time. This is represented schematically in figure 3.2. The technique originates from the nuclear magnetic resonance work of Bloembergen et al (1948). The situation for a simple two - level spin system can be represented by the following rate equation:

where n is the instantaneous difference in populations of the two energy levels; % is the Boltzmann - determined equilibrium difference of the populations. Signal recovery can be represented by the following equation:

S (t)= S L p - As„p, [ - t / T , ] .

where S^p is the signal amplitude at t =0 0 , AS is the difference in signal amplitudes at t=0

and t=oQ After a saturating pulse the pulse grows back in an exponential fashion. These equations are appropriate for an ESR transition of two levels and do not accommodate other ESR transitions or the presence of magnetic nuclei. Both of these result in complications, namely cross-relaxation and spectral diffusion which are time - dependent effects. For accurate Tj measurements the spin - lattice relaxation must be separated from the time - dependent phenomena. There are four basic conditions which need to be fulfilled to ensure this separation:

(1)Detected signals should exhibit a single exponential behaviour.

(2)The later part of the recovery curves should be taken to obtain the relaxation rate constants, as this data is the most reliable.

(3)Relaxation constants should be independent of the duration of microwave pumping. (4)They should also be independent of observing microwave power.

If the simplest case does not apply it may still be possible to extract rate constants by a number of strategies. For example by only fitting the latter part of the saturation curve which is the least susceptible to these time - dependent effects, or by use of a multiexponential fitting program which fits data over the entire time range.

f b

Fa

p . . ■ • ■ ■

•

.

O

(D" * _ 11

_

. ' 'O

0 , 0

P700

.

^Figure 3.1 6 A X - Ray Crystal Structure of Synechococcus

Figure 3.2

SP - saturating microwave pulse.

SE - spin echo. Tau is the time interval between the saturating and 90° microwave pulses. T is the time interval between the 90° and 180° microwave pulses.

The 2000 ns microwave pulse saturates the bulk magnetisation corresonding to a paramagnet so that the recovery of the bulk magnetisation can be measured. A microwave pulse with a duration of 16 ns is used to flip the recovering magnetisation through 90°. After a time interval, T, a microwave pulse with a duration of 32 ns is used to flip the magnetisation through 180,° so it lies along the y axis and can be detected. As the magnetisation relaxes microwave energy is released as a spin echo. It is the recovery of this spin echo which is determined spectroscopically.