Photophysics of Ionic Biochromophores

An ion with mass m, charge q and velocity v that encounters an electromagnetic field, composed of an electric field E and/or a magnetic field B, will experience the Lorentz force F = q(E + v × B). For both electrostatic parallel plate and cylindrical deflectors the potential difference V required to redirect an ion is dependent on the kinetic energy to charge ratio of the ion (Figs. 1.1 and 1.2). On the other hand, the magnitude of a magnetic field needed to deflect an ion along a radius of curvature r depends on the momentum to charge ratio of the ion (Fig. 1.3).

This chapter provides a brief introduction to biochromophores encountered in nature, and how their π-conjugated structures determine their excitation energies, i.e., their colour. Perturbations of electronic structure by a microenvironment such as water or charge sites are discussed. These may lead to a colour change (or modulation), depending on the character of the electronic transition. As detailed results for particular chromophores are presented in subsequent chapters, future challenges and new aspects within the research field are instead considered as the author sees them.

The description of the electronic structure of molecules in the excited state is usually more involved than the calculation of ground-state properties. The development of approaches to calculate optical properties of chromophores, including their specific interactions with a complex environment is a very active field of research. This chapter gives an overview of quantum mechanical methods and schemes to integrate them into a multi-scale description of extended systems that contain an optically active center. Special attention is paid to the problems and limitations of quantum methods that are commonly used to describe excited-state properties of biological chromophores.

This chapter combines recent advances in understanding the photophysics of the chromophore anion of the Green Fluorescent Protein (GFP) from the jellyfish Aequorea Victoria. GFP and its homologues are widely used for in vivo labeling in biology through their remarkable fluorescent properties. Besides long-timescale light emission, the GFP proteins also show an unusual diversity in terms of their non-radiative excited-state decay channels, including ultrafast conical intersection dynamics and light-driven electron transfer, where GFP acts as an electron donor in photochemical reactions. Knowledge of intrinsic properties of the GFP photoabsorbing molecular unit is a prerequisite in understanding the atomic-scale interactions that play a key role for the diverse functioning of these proteins. Here, we show how recent developments in action and photoelectron spectroscopy combined with state-of-the-art electronic structure theory provide valuable insights into photo-initiated quantum dynamics and enable to disclose mechanisms of multiple intrinsic excited-state decay channels in the bare GFP chromophore anion. When taken out of the protein, the deprotonated chromophore exhibits the ultrafast excited state dynamics, where non-radiative decay occurs on a (sub)picosecond timescale. Deactivation includes resonant electron emission and fast internal conversion followed by slow statistical decay in the vibrationally hot ground state. Remarkably, both electronic and nuclear excited-state decay channels may here efficiently compete with each other in spite of their inherently different intrinsic timescales. The reason behind this is an efficient coupling between the nuclear and electronic motion in the photo-initiated dynamics, where the energy may be transferred from nuclei to electrons and from electrons to nuclei mediated by specific vibrational modes. Prompt photodetachment occurs indirectly through vibrational autodetachment out of the first excited state within the energy range of the corresponding absorption band. Time-resolved transient photoelectron spectroscopy confirms the ultrafast decay of the excited-state population through internal conversion, which here proceeds through a conical intersection seam. We discuss the ways, by which the GFP proteins may use such efficient electron-to-nuclei coupling revealed in the intrinsic excited-state decay of their chromophore, to guide the photochemistry and photophysics upon which their functioning is based.

This chapter deals with measurements of fluorescence from electronically excited biomolecular ions where there are no interactions with an external environment. Biomolecules with no natural fluorophores are labelled with a dye for such experiments. First, some of the advantages, but also difficulties, of fluorescence spectroscopy compared to absorption spectroscopy are discussed. Extensive work has been done on the isolated dyes in characterising them with respect to their dispersed fluorescence spectra, excited-state lifetimes, and gas-phase Stokes shifts. After a brief introduction, results from experiments on dye-derivatised biomolecular ions that provide important information on folding/unfolding processes and local structural changes are presented. Examples included here are a model DNA duplex, the Trp-cage protein, polyproline peptides, and the cytochrome c heme protein. The chapter ends with a discussion on the oxyluciferin anion, the molecule responsible for light emission from fireflies where the electronic transition has charge-transfer character.

This chapter deals with gas-phase spectroscopy of protoporphyrin IX and heme ions, two important biochromophores in nature. These ions strongly absorb blue and green light, which accounts for e.g. the red colour of blood. We present absorption spectra of four-coordinate ferric heme cations at room temperature and in cold helium droplets, obtained in both cases from light-driven dissociation processes. These spectra serve as references for protein biospectroscopy and provide a natural testing ground for advanced quantum chemical modelling. The role of axial ligands bound to the iron centre, i.e., amino acids and nitric oxide, on the electronic structure of the porphyrin ring is discussed, and gas-phase spectra are compared to relevant protein ones. Spectroscopy on intact multiply charged protein anions with the heme prosthetic group is possible by monitoring the detachment of electrons triggered by light absorption. Similar experiments on protein cations rely on the absorption of too many photons for dissociation to be of practical use. This is illustrated from results on cytochrome c in vacuo. Time constants for dissociation and the corresponding dissociation channels obtained from storage-ring experiments are presented and discussed in the context of vibrational cooling within a heme protein cavity. Finally, we show the time spectrum for dissociation of photoexcited protoporphyrin IX anions, from which it is concluded that intersystem crossing to triplet states is in competition with internal conversion to the ground state. This is somewhat supported by spectroscopic characterisation of the long-lived states based on pump-probe experiments. Hence from one time spectrum (a one-laser experiment), triplet quantum yields can easily be estimated.

Optical spectroscopy has contributed enormously to our knowledge of the structure and dynamics of atoms and molecules and is now emerging as a cornerstone of the gas-phase methods available for investigating biomolecular ions. This chapter focuses on the UV and visible spectroscopy of peptide and protein ions stored in ion traps. First, we discuss experimental set-ups, de-excitation mechanisms following photo-excitation in electronically excited states and principles of action spectroscopy. Then, we report action spectra for different classes of gas-phase peptides and proteins. The optical activity of proteins in the near UV is directly related to the electronic structure and optical absorption of aromatic amino acids (Trp, Phe and Tyr). Some proteins also show absorption in the visible range due to the presence of a prosthetic group. Influence of protein charge state, formation of radical aromatic amino acids and solvation on proteins’ visible and UV spectra is discussed.

The electronic spectroscopy and the electronic excited state dynamics of the three protonated aromatic amino acids (PAAAs) phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp) are presented in this chapter. Ab-initio calculations combined with various experimental techniques such as coincidence experiments, pump-probe spectroscopy and photo-fragmentation of cold trapped ions have been implemented to work out the complex photophysical processes involved in these AAs. The results include photo-fragmentation spectra, excited-state lifetimes, full fragmentation patterns at fixed excitation energies and specific fragmentation channels as a function of the excitation energy. Fragmentation patterns depending on the electronic excited state are detailed.

Photodissociation studies on free complex protonated peptides and other biomolecular ions have long been limited to the UV wavelength range and longer wavelengths which are accessible by intense lasers. By interfacing tandem mass spectrometry with synchrotron beamlines, it is possible to overcome this limitation. We have thoroughly studied the interaction of vacuum ultraviolet (VUV) and soft X-ray photons with gas phase protonated peptides. Molecular fragmentation patterns, unobserved in conventional mass spectrometry, can be observed experimentally. Instead of relatively slow statistical fragmentation along the peptide backbone, much faster formation of fragment ions related to sidechains of aromatic amino acids is observed. The underlying process most likely involves fast charge migration. A previously unobserved dissociation scheme, in which photoabsorption leads to a fast loss of a tyrosine side chain can be observed for the VUV and soft X-ray range. This loss process leads to the formation of a residual peptide that is remarkably cold internally.