Ultrafast spectroscopy of biological photoreceptors

We review recent new insights on reaction dynamics of photoreceptors proteins gained from ultrafast spectroscopy. In Blue Light sensing Using FAD (BLUF) domains, a hydrogen-bond rearrangement around the flavin chromophore proceeds through a radical-pair mechanism, by which light-induced electron and proton transfer from the protein to flavin result in rotation of a conserved glutamine that switches the hydrogen bond network. Femtosecond infrared spectroscopy has shown that in photoactive yellow protein (PYP), breaking of a hydrogen bond that connects the p-coumaric acid chromophore to the backbone is crucial for trans–cis isomerization and successful entry into the photocycle. Furthermore, isomerization reactions of phycocyanobilin in phytochrome and retinal in the rhodopsins have been revealed in detail through application of femtosecond infrared and femtosecond-stimulated Raman spectroscopy.

Introduction

Photoreceptor proteins are exceptionally interesting objects of study, not only in relation to their biological function but also because they can be triggered by a short flash of light, allowing the study of functional protein dynamics over a wide span of timescales. The initial events after photon absorption typically occur in less than 1 ns. As essentially all elementary physical and chemical transformations in biology inherently are ultrafast but often limited by slow diffusional motions that obscure their true dynamics, photoreceptor proteins afford a unique avenue to learn more about the nature of processes such as electron or proton transfer, making or breaking of chemical bonds and motions of small molecular groups.

The past decade has witnessed the discovery and characerization of a large number of novel photoreceptors, most notably the proteorhodopsins [1], the Light, Oxygen or Voltage (LOV) domains [2, 3, 4] and the Blue Light sensing Using FAD (BLUF) domains [5]. BLUF and LOV domains are of special interest as they bind a flavin rather than an isomerizing cofactor, making their photochemistry radically different from that of ‘traditional’ photoreceptors such as the rhodopsins, phytochromes and xanthopsins [6]. The flavin photochemistry enables a strict separation regarding the roles of cofactor and protein: isomerizing cofactors exhibit isomerization and twisting reactions in solution and even in vacuo [7^•], rendering the influence and catalyzing properties of the protein difficult to assess. By contrast, flavins need partner molecules to react. Thus, flavin-based receptors pose us with new concepts and opportunities to understand how light absorption may be coupled to biological sensory function through efficient and selective photochemistry.

While ultrafast spectroscopy at visible and ultraviolet (UV) wavelengths is quite suitable to characterize transient flavin intermediates in LOV and BLUF domains, assessing the structure of isomerizing cofactors while they react remains impossible with this method. This point was explicitly demonstrated in a study on bacteriorhodopsin (bR), where a number of femtosecond optical signals could be well described by a nonreactive model for the retinal chromophore [8]. To determine dynamic structures of chromophores and the interaction with nearby side chains, ultrafast vibrational spectroscopic techniques need to be applied. Here, we review the recent new insights into the early events that occur in biological photoreceptors. We address the photochemistry and the light activation mechanism of BLUF domains and will treat the isomerization mechanisms of photoactive yellow protein, phytochrome and rhodopsins as determined by the novel vibrational techniques, femtosecond infrared (femtoIR) spectroscopy, and femtosecond-stimulated Raman spectroscopy (FSRS).