Single Amino Acid Mutation Decouples Photochemistry of the BLUF Domain from the Enzymatic Function of OaPAC and Drives the Enzyme to a Switched-on State.

Photoactivated adenylate cyclases (PACs) are light-activated enzymes that combine a BLUF (blue-light using flavin) domain and an adenylate cyclase domain that are able to increase the levels of the important second messenger cAMP (cyclic adenosine monophosphate) upon blue-light excitation. The light-induced changes in the BLUF domain are transduced to the adenylate cyclase domain via a mechanism that has not yet been established. One critical residue in the photoactivation mechanism of BLUF domains, present in the vicinity of the flavin is the glutamine amino acid close to the N5 of the flavin. The role of this residue has been investigated extensively both experimentally and theoretically. However, its role in the activity of the photoactivated adenylate cyclase, OaPAC has never been addressed. In this work, we applied ultrafast transient visible and infrared spectroscopies to study the photochemistry of the Q48E OaPAC mutant. This mutation altered the primary electron transfer process and switched the enzyme into a permanent 'on' state, able to increase the cAMP levels under dark conditions compared to the cAMP levels of the dark-adapted state of the wild-type OaPAC. Differential scanning calorimetry measurements point to a less compact structure for the Q48E OaPAC mutant. The ensemble of these findings provide insight into the important elements in PACs and how their fine tuning may help in the design of optogenetic devices.

Breaking Barriers in Ultrafast Spectroscopy and Imaging Using 100 kHz Amplified Yb-Laser Systems.

ConspectusUltrafast spectroscopy and imaging have become tools utilized by a broad range of scientists involved in materials, energy, biological, and chemical sciences. Commercialization of ultrafast spectrometers including transient absorption spectrometers, vibrational sum frequency generation spectrometers, and even multidimensional spectrometers have put these advanced spectroscopy measurements into the hands of practitioners originally outside the field of ultrafast spectroscopy. There is now a technology shift occurring in ultrafast spectroscopy, made possible by new Yb-based lasers, that is opening exciting new experiments in the chemical and physical sciences. Amplified Yb-based lasers are not only more compact and efficient than their predecessors but also, most importantly, operate at many times the repetition rate with improved noise characteristics in comparison to the previous generation of Ti:sapphire amplifier technologies. Taken together, these attributes are enabling new experiments, generating improvements to long-standing techniques, and affording the transformation of spectroscopies to microscopies. This Account aims to show that the shift to 100 kHz lasers is a transformative step in nonlinear spectroscopy and imaging, much like the dramatic expansion that occurred with the commercialization of Ti:sapphire laser systems in the 1990s. The impact of this technology will be felt across a great swath of scientific communities. We first describe the technology landscape of amplified Yb-based laser systems used in conjunction with 100 kHz spectrometers operating with shot-to-shot pulse shaping and detection. We also identify the range of different parametric conversion and supercontinuum techniques which now provide a path to making pulses of light optimal for ultrafast spectroscopy. Second, we describe specific instances from our laboratories of how the amplified Yb-based light sources and spectrometers are transformative. For multiple probe time-resolved infrared and transient 2D IR spectroscopy, the gain in temporal span and signal-to-noise enables dynamical spectroscopy measurements from femtoseconds to seconds. These gains widen the applicability of time-resolved infrared techniques across a range of topics in photochemistry, photocatalysis, and photobiology as well as lower the technical barriers to implementation in a laboratory. For 2D visible spectroscopy and microscopy with white light, as well as 2D IR imaging, the high repetition rates of these new Yb-based light sources allow one to spatially map 2D spectra while maintaining high signal-to-noise in the data. To illustrate the gains, we provide examples of imaging applications in the study of photovoltaic materials and spectroelectrochemistry.

Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy.

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule.

Excited-state charge separation in the photochemical mechanism of the light-driven enzyme protochlorophyllide oxidoreductase.

The unique light-driven enzyme protochlorophyllide oxidoreductase (POR) is an important model system for understanding how light energy can be harnessed to power enzyme reactions. The ultrafast photochemical processes, essential for capturing the excitation energy to drive the subsequent hydride- and proton-transfer chemistry, have so far proven difficult to detect. We have used a combination of time-resolved visible and IR spectroscopy, providing complete temporal resolution over the picosecond-microsecond time range, to propose a new mechanism for the photochemistry. Excited-state interactions between active site residues and a carboxyl group on the Pchlide molecule result in a polarized and highly reactive double bond. This so-called "reactive" intramolecular charge-transfer state creates an electron-deficient site across the double bond to trigger the subsequent nucleophilic attack of NADPH, by the negatively charged hydride from nicotinamide adenine dinucleotide phosphate. This work provides the crucial, missing link between excited-state processes and chemistry in POR. Moreover, it provides important insight into how light energy can be harnessed to drive enzyme catalysis with implications for the design of light-activated chemical and biological catalysts.