Selected photochemical and photophysical parameters of flavin mononucleotide (FMN) have been examined under conditions in which FMN is (1) solvated in a buffered aqueous solution, and (2) encased in a protein likewise solvated in a buffered aqueous solution. The latter was achieved using the so-called "mini Singlet Oxygen Generator" (miniSOG), an FMN-containing flavoprotein engineered from Arabidopsis thaliana phototropin 2. Although FMN is a reasonably good singlet oxygen photosensitizer in bulk water (ϕΔ = 0.65 ± 0.04), enclosing FMN in this protein facilitates photoinitiated electron-transfer reactions (Type-I chemistry) at the expense of photosensitized singlet oxygen production (Type-II chemistry) and results in a comparatively poor yield of singlet oxygen (ϕΔ = 0.030 ± 0.002). This observation on the effect of the local environment surrounding FMN is supported by a host of spectroscopic and chemical trapping experiments. The results of this study not only elucidate the behavior of miniSOG but also provide useful information for the further development of well-characterized chromophores suitable for use as intracellular sensitizers in mechanistic studies of reactive oxygen species.

Singlet molecular oxygen, O(2)(a(1)Delta(g)), is an intermediate in a variety of oxygenation reactions. The reactivity of singlet oxygen in a given system is influenced, in part, by competitive solvent-dependent channels that deactivate singlet oxygen in a nonradiative process. It has long been considered that these deactivation channels depend only slightly on temperature. This conclusion has been incorporated into the accepted empirically derived model of electronic-to-vibrational energy transfer used to account for the effect of solvent on the lifetime of singlet oxygen, tau(Delta). The current study reveals that tau(Delta), in fact, can depend quite significantly on temperature in certain solvents (e.g., D(2)O and benzene-d(6)). These results can have practical ramifications in studies of singlet oxygen reactivity. From a fundamental perspective, these data indicate that aspects of the model for nonradiative deactivation of singlet oxygen need to be re-evaluated.

Singlet molecular oxygen, O(2)(a(1)Δ(g)), can influence many processes pertinent to the function of biological systems, including events that result in cell death. Many of these processes involve a reaction between singlet oxygen and a given amino acid in a protein. As a result, the behavior of that protein can change, either because of a structural alteration and/or a direct modification of an active site. Surprisingly, however, little is known about rate constants for reactions between singlet oxygen and amino acids when the latter are in a protein. In this report, we demonstrate using five separate proteins, each containing only a single tryptophan residue, that the rate constant for singlet oxygen reaction with tryptophan depends significantly on the position of this amino acid in the protein. Most importantly, the reaction rate constant depends not only on the accessibility of the tryptophan residue to oxygen, but also on factors that characterize the local molecular environment of the tryptophan in the protein. The fact that the local protein environment can either appreciably inhibit or accelerate the reaction of singlet oxygen with a given amino acid can have significant ramifications for singlet-oxygen-mediated events that perturb cell function.

Linear and nonlinear spectroscopic parameters of flavin mononucleotide, FMN, have been examined both experimentally and computationally under conditions in which FMN is (1) solvated in a buffered aqueous solution, and (2) encased in a protein that is likewise solvated in a buffered aqueous solution. The latter was achieved using "miniSOG" which is an FMN-containing protein engineered from Arabidopsis thaliana phototropin 2. Although it is reasonable to expect that the encasing protein could have an appreciable effect, certainly on the nonlinear two-photon absorption cross section, we find that replacing the dynamic aqueous environment with the more static protein environment does little to influence the spectroscopic properties of FMN. The experimental and computational studies are consistent in this regard, and this agreement indicates that comparatively high-level computational methods can indeed be used with success on large chromophores with a complicated local environment. The results of the present study facilitate the much-needed development of well-characterized and readily-controlled chromophores suitable for use as intracellular sensitizers and fluorophores.

Controlling and quantifying the photosensitized production of singlet oxygen are key aspects in mechanistic studies of oxygen-dependent photoinitiated cell death. In this regard, the commonly accepted practice of using intracellular photosensitizers is, unfortunately, plagued by problems that include the inability to accurately (1) quantify the sensitizer concentration in the irradiated domain and (2) control the local environment that influences light delivery and sensitizer photophysics. However, capitalizing on the fact that singlet oxygen produced outside a cell is also cytotoxic, many of these problems can be avoided with the use of an extracellular sensitizer. For the present study, a hydrophilic dendrimer-encased membrane-impermeable sensitizer was used to generate an extracellular population of singlet oxygen upon spatially localized two-photon irradiation. Through the use of this sensitizer and this approach, it is now possible to better control the singlet oxygen dose in microscope-based time- and space-resolved single cell experiments. Thus, we provide a solution to a limiting problem in mechanistic studies of singlet-oxygen-mediated cell death.