Copyright © 2008 by Kathryn Thompson and Juan Rodriguez 1Topic 7: The Behavior of Photo-Excited Molecules: Biological Actions and Biotechnological Applications 7.1 INTRODUCTION Topic 6 gave us a foundation for understanding the process of light absorption in organic molecules. We now know that when a photon strikes a molecule, one of its electrons can absorb the energy and be promoted to an excited state. What happens next? Does the molecule retain that energy indefinitely? Is it converted into other forms? Here we discuss the fundamental processes that ensue from light absorption, and their relevance to biology as well as important biotechnological applications. 7.2 KASHA’S RULE In 1953, Michael Kasha proposed that no matter how much light energy a molecule absorbs, it undergoes a rapid transition to the first excited state. Since the transition occurs typically in less than one picosecond, we can assume that the molecule will always be effectively excited to the first excited state regardless of the energy carried by the photon. The process is sown in Fig. 1. Furthermore, the transition from higher excited states to the first excited state usually emits no light, and is thus categorized as a radiationless transition. Figure 1. If a molecule is excited to an energy level higher than the first excited state, it undergoes a radiationless transition to this state, releasing excess heat into its environment. In a radiationless transition, the electronic energy difference is converted into vibrational energy. This results in a higher internal temperature for the molecule, but it quickly (within 10 ps) returns to ambient temperature by releasing heat into the surrounding medium. From the first excited state, the molecule can proceed in one of many ways. In this chapter we will examine six possible outcomes for an excited molecule: radiationless transition to the ground state; fluorescence; intersystem crossing; phosphorescence; excitation transfer to another nearby molecule; and charge transfer to another nearby molecule. We will also discuss several biological systems and technologies that take advantage of these photophysical phenomena.Copyright © 2008 by Kathryn Thompson and Juan Rodriguez 27.3 RADIATIONLESS TRANSITION TO THE GROUND STATE A radiationless transition to the ground state is essentially the same as a radiationless transition from higher excited states to the first excited state, described in section 7.2. The only difference is that the molecule now transitions from the first excited state to the ground state. No photons are emitted, and the excess energy is converted to heat, which is quickly released into the surrounding medium. Figure 2. Radiationless transfer to the ground state This process usually takes place on a nanosecond timescale, substantially longer than radiationless transitions from higher excited states. Some molecules — such as metalloporphyrins like hemes and cytochromes — decay to the ground state very quickly, within a few picoseconds. This fast decay is due to the presence of iron in the center of these molecules, which creates additional electronic states between the first excited state and the ground state that facilitate the decay. Temperatures can reach 200oC inside these molecules when the energy is converted to heat, but the heat is dissipated so quickly that the molecules suffer no damage. 7.4 FLUORESCENCE In fluorescence, as shown in Fig.2, the molecule begins in an excited state with no net spin, as all the spins of the electrons cancel each other out. Within nanoseconds, a photon is emitted and the molecule returns to ground state. The net spin remains zero. The energy lost by the electron inside the molecule is entirely converted into photon energy. Figure 3. Fluorescence. Note that the photon emitted has the same energy as the energy difference between the excited and ground statesCopyright © 2008 by Kathryn Thompson and Juan Rodriguez 3You are familiar with fluorescence if you have ever seen your clothes “glow” under a black light. Most light-colored fabrics absorb some blue light, so they tend to naturally appear yellowish, even when clean. To make them look whiter, we wash them with detergents that contain “whiteners,” small fluorescent molecules that adhere to fabric. Illuminating detergent with ultraviolet light causes it to fluoresce bright blue. The added blue light emanating from the detergent molecules causes the fabric to appear whiter. When isolated from their natural environment, most biological molecules exhibit a significant amount of fluorescence. For example, if you grind leaves from a green plant and suspend the particles in solution so that the pigment molecules are isolated from one another, the solution will fluoresce red-orange under ultraviolet light. Not all biomolecules do this, however; exceptions include heme and the cytochromes, which undergo a radiationless transition before they have a chance to fluoresce. In vivo, however, most biological molecules display modest fluorescence, particularly within the visible range. Either the fluorescence does occur but is quickly reabsorbed by surrounding molecules, or the molecule transfers the energy or an electron to a nearby molecule. An exception to the lack of in vivo fluorescence occurs in association with the rare genetic disease porphyria. One form of the disease is caused by an enzyme that fails to catalyze the binding of iron to the porphyrin ring of heme. The porphyrins build up over time in the teeth and bones, which, if irradiated with UV light display a reddish fluorescence. 7.5 INTERSYSTEM CROSSING In intersystem crossing, the molecule begins in an excited state with a net spin of 0 because all electron spins cancel one another. After a time, typically 10 nsec or longer, it ends in an excited state with a net spin of 1 (i.e. a spin angular momentum with a magnitude equal to one h or π2h . This end state is called a triplet state because of a quantum mechanical effect that produces three possible states with different angular momentum orientations†. Figure 4. Intersystem crossing. 11† In general, quantum mechanical systems are allowed to change their angular momentum, but only in increments of h . A system n electron with a spin magnitude of system with a spin angular momentum can change its direction, but only in orientations whose angular momenta differ by magnitude of h can two