Orange Carotenoid Protein (OCP) is a unique photoactive protein containing a carotenoid as the photo-responsive chromophore, involved in cyanobacterial photoprotection. The photo-activation of OCP begins with the picosecond evolution of the ketocarotenoid excited-state levels and involves structural changes occurring up to second time scale, ultimately leading to the active red OCP with a quantum yield of about 0.2%. There is a longstanding debate on the origin of the red OCP's low quantum yield and the intermediates that control it. Additionally, reports published so far implicitly assume that the absorption of only one photon is enough to initiate complete photoconversion to the active OCP form.
The major challenge in deducing the photoactivation mechanism is creating a uniform explanation for both single-pulse excitation experiments and continuous light irradiation experiments. We studied the photodynamics of different OCPs and carotenoids using time-resolved X-ray scattering and Visible-NIR transient absorption spectroscopy, considering variables such as concentration, His-tagging, excitation pulse power, and wavelength. Additionally, we performed nanosecond to second time resolved transient absorption experiments coupled with stationary irradiation light to verify the single-photon hypothesis.
Our experiments showed a light intensity dependence of the photo-activation yield. These results clearly demonstrate that single-photon absorption cannot explain the observed dynamics of OCP under biological irradiation conditions encountered in vivo. We will then discuss here the entire photo-activation mechanism of OCP, from the femtosecond to second time scale, with a special focus on the light intensity response.