Photocycling Proteins

Upon absorption of a photon, the photocycling protein undergoes a series of conformational changes generating intermediate state(s); one of these states is the “active” state that will start signal transmission. The last intermediate state is driven back to the original state of the protein, by either a thermal process, or a second absorbed photon of different wavelength. The primary event in the photoreceptive process is the structural change of the chromophore (isomerization) to which the protein adapts. It occurs within a few picoseconds after the absorption of a photon, and this is one of the fastest biological processes in nature. The whole photocycle is very fast (order of microseconds or less), hence the intracellular response is immediately reset so that the system is prepared for a new light signal, and algae must respond rapidly on a time scale of milliseconds to seconds as environmental conditions change or as they change position relative to their static surroundings. A photoreceptor protein capable of photocycling is mandatory for algae whose photoreceptive systems are an integral part of the cell body. This localization would not allow the continuous recovery of the exhausted photoreceptive proteins without interfering with a continuous and immediate response of the alga cell to the light.

Sensitivity
The ability to perceive and adapt to changing light conditions is critical to the life and growth of photosynthetic microorganisms. Light quality and quantity varies diurnally, seasonally, and with latitude, and is influenced by cloud conditions and atmospheric absorption (e.g., pollution). Competition for light in aquatic environment may be particularly fierce because of shading among the different organisms and the rapid absorption of light in the water column. Illumination of the surface layers varies with place, time, and conditions depending on the intensity of light penetrating the surface and upon the transparency of the water. Hence, detecting light as low as possible (i.e., a single photon) becomes an adaptive advantage, because a photosynthetic organism in dim light can obtain more metabolic energy if it is able to move to more lighted and suitable areas.

For detecting the direction of light of a specific spectral range, a photoreceptor demands a high packing density of chromophore molecules organized in a lattice structure, with high absorption cross-section of the chromophore, that is, high probability of photon absorption by the chromophore, and very low dark noise. For detecting patterns of light, the number and location of photoreceptors having fixed size and exposure time must be viewed according to the pattern of motion of algae. For transmitting the detected signal a photoreceptor must generate a potential difference, or a current.

The most investigated photoreception system is that of Chlamydomonas. It consists of a patch of rhodopsin-like proteins in the plasma membrane (Type I). The packing density of these molecules appears to be about 20,000–30,000 µm^-2 of membrane, with a molar absorption coefficient 1 of 40,000–60,000 M^-1 cm^-1 and a dark noise (see later) approximately equal to zero. The number of embedded molecules per square micrometer of membrane, the absorption cross-section, and the dark noise are at the best of theoretical limits. Nevertheless, the fraction of photon absorbed from a single layer of these molecules is less than 0.05% (each layer contributes approximately to 0.005 OD).

Let us calculate how many photons this simplest but real photoreceptive system can absorb (Type I). In a sunny day about 10¹⁸ photons per square meter per second per nanometer are emitted by the sun (in a cloudy day the number of photons lowers to 10¹⁷). As algae dwell in an aquatic environment we have to consider absorption and reflection effects of the water, and we lower this figure to 10¹⁷ photons per second per nanometer (in a cloudy day the number of photons lowers to 10¹⁶). This means that a photoreceptor of 1 µm² can catch at most 10⁵ photons per second per nanometer, that is, 10⁷ photons in its 100 nm absorbance window in the sunniest day. As only 0.05% of the incident radiation will be effectively absorbed by a single-layer photoreceptor, the amount of photons lowers to about 10³ photons per second. The true signal that the algae should discriminate results from the difference between the number of photons absorbed by the photoreceptor when it is illuminated (for about 400 msec) and the number of photons absorbed by the photoreceptor when it is shaded by a screen such as the eyespot (for about 600 msec). In the case of the sunniest day the highest signal is lower than 100 photons per second, which lowers to about 10 photons per second in a cloudy day. Even in the sunniest day the number of photons absorbed for detecting light direction is very low. As this photoreceptor does not form any image, but detects only light intensity, this amount of photons is enough; however, this photoreceptor must posses a very low threshold and a very low or negligible dark noise.

It has been demonstrated that not only single photons induce transient direction changes but also fluence rates as low as 1 photon cell^-1 sec^-1 can actually lead to a persistent orientation in Chlamydomonas (Chlorophyceae).

Strategies have been evolved to increase the sensitivity of a photoreceptor, as stacking-up many pigment-containing membranes in the direction of the light path (Euglena gracilis is a wonderful example of this solution) or exploiting the reflecting properties of the eyespot as in Chlamydomonas and in different species of dinoflagellates. The effectiveness of the multilayer strategy has been experimentally tested by Robert R. Birge of the W.M. Keck Center of the Syracuse University (personal communication). The absorption value recorded on a preparation of precisely ordered rhodopsin- like proteins multilayers was about 95% of the incident light, very close to the absorption value recorded on the photoreceptor crystal of Euglena, which consists of more than 100 layers of proteins.