Photoinduced Electron Transfer from a 1,4,5,6-Tetrahydro Nicotinamide Adenine Dinucleotide (Phosphate) Analogue to Oxidized Flavin in an Ene-Reductase Flavoenzyme.

Recent reports have described the use of ene-reductase flavoenzymes to catalyze non-natural photochemical reactions. These studies have focused on using reduced flavoenzyme, yet oxidized flavins have superior light harvesting properties. In a binary complex of the oxidized ene-reductase pentaerythritol tetranitrate reductase with the nonreactive nicotinamide coenzyme analogs 1,4,5,6-tetrahydro NAD(P)H, visible photoexcitation of the flavin mononucleotide (FMN) leads to one-electron transfer from the NAD(P)H4 to FMN, generating a NAD(P)H4 cation radical and anionic FMN semiquinone. This electron transfer occurs in ∼1 ps and appears to kinetically outcompete reductive quenching from aromatic residues in the active site. Time-resolved infrared measurements show that relaxation processes appear to be largely localized on the FMN and the charge-separated state is short-lived, with relaxation, presumably via back electron transfer, occurring over ∼3-30 ps. While this demonstrates the potential for non-natural photoactivity, useful photocatalysis will likely require longer-lived excited states, which may be accessible by enzyme engineering and/or a judicious choice of substrate.

Not All 3MC States Are the Same: The Role of 3MCcis States in the Photochemical N∧N Ligand Release from [Ru(bpy)2(N∧N)]2+ Complexes.

Ruthenium(II) complexes feature prominently in the development of agents for photoactivated chemotherapy; however, the excited-state mechanisms by which photochemical ligand release operates remain unclear. We report here a systematic experimental and computational study of a series of complexes [Ru(bpy)2(N∧N)]2+ (bpy = 2,2'-bipyridyl; N∧N = bpy (1), 6-methyl-2,2'-bipyridyl (2), 6,6'-dimethyl-2,2'-bipyridyl (3), 1-benzyl-4-(pyrid-2-yl)-1,2,3-triazole (4), 1-benzyl-4-(6-methylpyrid-2-yl)-1,2,3-triazole (5), 1,1'-dibenzyl-4,4'-bi-1,2,3-triazolyl (6)), in which we probe the contribution to the promotion of photochemical N∧N ligand release of the introduction of sterically encumbering methyl substituents and the electronic effect of replacement of pyridine by 1,2,3-triazole donors in the N∧N ligand. Complexes 2 to 6 all release the ligand N∧N on irradiation in acetonitrile solution to yield cis-[Ru(bpy)2(NCMe)2]2+, with resultant photorelease quantum yields that at first seem counter-intuitive and span a broad range. The data show that incorporation of a single sterically encumbering methyl substituent on the N∧N ligand (2 and 5) leads to a significantly enhanced rate of triplet metal-to-ligand charge-transfer (3MLCT) state deactivation but with little promotion of photoreactivity, whereas replacement of pyridine by triazole donors (4 and 6) leads to a similar rate of 3MLCT deactivation but with much greater photochemical reactivity. The data reported here, discussed in conjunction with previously reported data on related complexes, suggest that monomethylation in 2 and 5 sterically inhibits the formation of a 3MCcis state but promotes the population of 3MCtrans states which rapidly deactivate 3MLCT states and are prone to mediating ground-state recovery. On the other hand, increased photochemical reactivity in 4 and 6 seems to stem from the accessibility of 3MCcis states. The data provide important insights into the excited-state mechanism of photochemical ligand release by Ru(II) tris-bidentate complexes.

Investigating radical pair reaction dynamics of B12 coenzymes 1: Transient absorption spectroscopy and magnetic field effects.

B12 coenzymes are vital to healthy biological function across nature. They undergo radical chemistry in a variety of contexts, where spin-correlated radical pairs can be generated both thermally and photochemically. Owing to the unusual magnetic properties of B12 radical pairs, however, most of the reaction and spin dynamics occur on a timescale (picoseconds-nanoseconds) that cannot be resolved by most measurement techniques. Here, we describe a method that combines femtosecond transient absorption spectroscopy with magnetic field exposure, which enables the direct scrutiny of such rapid processes. This approach should provide a means by which to investigate the apparently profound effect protein environments have on the generation and reactivity of B12 radical pairs.

An unusual light-sensing function for coenzyme B12 in bacterial transcription regulator CarH.

Coenzyme B12 is one of the most complex cofactors found in nature and synthesized de novo by certain groups of bacteria. Although its use in various enzymatic reactions is well characterized, only recently an unusual light-sensing function has been ascribed to coenzyme B12. It has been reported that the coenzyme B12 binding protein CarH, found in the carotenoid biosynthesis pathway of several thermostable bacteria, binds to the promoter region of DNA and suppresses transcription. To overcome the harmful effects of light-induced damage in the cells, CarH releases DNA in the presence of light and promotes transcription and synthesis of carotenoids, thereby working as a photoreceptor. CarH is able to achieve this by exploiting the photosensitive nature of the CoC bond between the adenosyl moiety and the cobalt atom in the coenzyme B12 molecule. Extensive structural and spectroscopy studies provided a mechanistic understanding of the molecular basis of this unique light-sensitive reaction. Most studies on CarH have used the ortholog from the thermostable bacterium Thermus thermophilus, due to the ease with which it can be expressed and purified in high quantities. In this chapter we give an overview of this intriguing class of photoreceptors and report a step-by-step protocol for expression, purification and spectroscopy experiments (both static and time-resolved techniques) employed in our laboratory to study CarH from T. thermophilus. We hope the contents of this chapter will be of interest to the wider coenzyme B12 community and apprise them of the potential and possibilities of using coenzyme B12 as a light-sensing probe in a protein scaffold.

How Photoactivation Triggers Protochlorophyllide Reduction: Computational Evidence of a Stepwise Hydride Transfer during Chlorophyll Biosynthesis.

The photochemical reaction catalyzed by enzyme protochlorophyllide oxidoreductase (POR), a rare example of a photoactivated enzyme, is a crucial step during chlorophyll biosynthesis and involves the fastest known biological hydride transfer. Structures of the enzyme with bound substrate protochlorophyllide (PChlide) and coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) have recently been published, opening up the possibility of using computational approaches to provide a comprehensive understanding of the excited state chemistry. Herein, we propose a complete mechanism for the photochemistry between PChlide and NADPH based on density functional theory (DFT) and time-dependent DFT calculations that is consistent with recent experimental data. In this multi-step mechanism, photoexcitation of PChlide leads to electron transfer from NADPH to PChlide, which in turn facilitates hydrogen atom transfer by weakening the breaking C-H bond. This work rationalizes how photoexcitation facilitates hydride transfer in POR and has more general implications for biological hydride transfer reactions.

Photochemistry of Heteroleptic 1,4,5,8-Tetraazaphenanthrene- and Bi-1,2,3-triazolyl-Containing Ruthenium(II) Complexes.

Diimine metal complexes have significant relevance in the development of photodynamic therapy (PDT) and photoactivated chemotherapy (PACT) applications. In particular, complexes of the TAP ligand (1,4,5,8-tetraazaphenanthrene) are known to lead to photoinduced oxidation of DNA, while TAP- and triazole-based complexes are also known to undergo photochemical ligand release processes relevant to PACT. The photophysical and photochemical properties of heteroleptic complexes [Ru(TAP)n(btz)3-n]2+ (btz = 1,1'-dibenzyl-4,4'-bi-1,2,3-triazolyl, n = 1 (1), 2 (2)) have been explored. Upon irradiation in acetonitrile, 1 displays analogous photochemistry to that previously observed for [Ru(bpy)(btz)2]2+ (bpy = 2,2'-bipyridyl) and generates trans-[Ru(TAP)(btz)(NCMe)2]2+ (5), which has been crystallographically characterized, with the observation of the ligand-loss intermediate trans-[Ru(TAP)(κ2-btz)(κ1-btz)(NCMe)]2+ (4). Complex 2 displays more complicated photochemical behavior with not only preferential photorelease of btz to form cis-[Ru(TAP)2(NCMe)2]2+ (6) but also competitive photorelease of TAP to form 5. Free TAP is then taken up by 6 to form [Ru(TAP)3]2+ (3) with the proportion of 5 and 3 observed to progressively increase during prolonged photolysis. Data suggest a complex set of reversible photochemical ligand scrambling processes in which 2 and 3 are interconverted. Computational DFT calculations have enabled optimization of geometries of the pro-trans 3MCcis states with repelled btz or TAP ligands crucial for the formation of 5 from 1 and 2, respectively, lending weight to recent evidence that such 3MCcis states play an important mechanistic role in the rich photoreactivity of Ru(II) diimine complexes.

Photocatalysis as the 'master switch' of photomorphogenesis in early plant development.

Enzymatic photocatalysis is seldom used in biology. Photocatalysis by light-dependent protochlorophyllide oxidoreductase (LPOR)-one of only a few natural light-dependent enzymes-is an exception, and is responsible for the conversion of protochlorophyllide to chlorophyllide in chlorophyll biosynthesis. Photocatalysis by LPOR not only regulates the biosynthesis of the most abundant pigment on Earth but it is also a 'master switch' in photomorphogenesis in early plant development. Following illumination, LPOR promotes chlorophyll production, plastid membranes are transformed and the photosynthetic apparatus is established. Given these remarkable, light-induced pigment and morphological changes, the LPOR-catalysed reaction has been extensively studied from catalytic, physiological and plant development perspectives, highlighting vital, and multiple, cellular roles of this intriguing enzyme. Here, we offer a perspective in which the link between LPOR photocatalysis and plant photomorphogenesis is explored. Notable breakthroughs in LPOR structural biology have uncovered the structural-mechanistic basis of photocatalysis. These studies have clarified how photon absorption by the pigment protochlorophyllide-bound in a ternary LPOR-protochlorophyllide-NADPH complex-triggers photocatalysis and a cascade of complex molecular and cellular events that lead to plant morphological changes. Photocatalysis is therefore the master switch responsible for early-stage plant development and ultimately life on Earth.

Dual role of the active site 'lid' regions of protochlorophyllide oxidoreductase in photocatalysis and plant development.

Protochlorophyllide oxidoreductase (POR) catalyses reduction of protochlorophyllide (Pchlide) to chlorophyllide, a light-dependent reaction of chlorophyll biosynthesis. POR is also important in plant development as it is the main constituent of prolamellar bodies in etioplast membranes. Prolamellar bodies are highly organised, paracrystalline structures comprising aggregated oligomeric structures of POR-Pchlide-NADPH complexes. How these oligomeric structures are formed and the role of Pchlide in oligomerisation remains unclear. POR crystal structures highlight two peptide regions that form a 'lid' to the active site, and undergo conformational change on binding Pchlide. Here, we show that Pchlide binding triggers formation of large oligomers of POR using size exclusion chromatography. A POR 'octamer' has been isolated and its structure investigated by cryo-electron microscopy at 7.7 Å resolution. This structure shows that oligomer formation is most likely driven by the interaction of amino acid residues in the highly conserved lid regions. Computational modelling indicates that Pchlide binding stabilises exposure of hydrophobic surfaces formed by the lid regions, which supports POR dimerisation and ultimately oligomer formation. Studies with variant PORs demonstrate that lid residues are involved in substrate binding and photocatalysis. These highly conserved lid regions therefore have a dual function. The lid residues position Pchlide optimally to enable photocatalysis. Following Pchlide binding, they also enable POR oligomerisation - a process that is reversed through subsequent photocatalysis in the early stages of chloroplast development.

Photochemical Mechanism of Light-Driven Fatty Acid Photodecarboxylase.

Fatty acid photodecarboxylase (FAP) is a promising target for the production of biofuels and fine chemicals. It contains a flavin adenine dinucleotide cofactor and catalyzes the blue-light-dependent decarboxylation of fatty acids to generate the corresponding alkane. However, little is known about the catalytic mechanism of FAP, or how light is used to drive enzymatic decarboxylation. Here, we have used a combination of time-resolved and cryogenic trapping UV-visible absorption spectroscopy to characterize a red-shifted flavin intermediate observed in the catalytic cycle of FAP. We show that this intermediate can form below the "glass transition" temperature of proteins, whereas the subsequent decay of the species proceeds only at higher temperatures, implying a role for protein motions in the decay of the intermediate. Solvent isotope effect measurements, combined with analyses of selected site-directed variants of FAP, suggest that the formation of the red-shifted flavin species is directly coupled with hydrogen atom transfer from a nearby active site cysteine residue, yielding the final alkane product. Our study suggests that this cysteine residue forms a thiolate-flavin charge-transfer species, which is assigned as the red-shifted flavin intermediate. Taken together, our data provide insights into light-dependent decarboxylase mechanisms catalyzed by FAP and highlight important considerations in the (re)design of flavin-based photoenzymes.

Photocycle of Cyanobacteriochrome TePixJ.

Due to the recent advances in X-ray free electron laser techniques, bilin-containing cyanobacteriochrome photoreceptors have become prime targets for the ever-expanding field of time-resolved structural biology. However, to facilitate these challenging studies, it is essential that the time scales of any structural changes during the photocycles of cyanobacteriochromes be established. Here, we have used visible and infrared transient absorption spectroscopy to probe the photocycle of a model cyanobacteriochrome system, TePixJ. The kinetics span multiple orders of magnitude from picoseconds to seconds. Localized changes in the bilin binding pocket occur in picoseconds to nanoseconds, followed by more large-scale changes in protein structure, including formation and breakage of a second thioether linkage, in microseconds to milliseconds. The characterization of the entire photocycle will provide a vital frame of reference for future time-resolved structural studies of this model photoreceptor.

Ultrafast Vibrational Energy Transfer between Protein and Cofactor in a Flavoenzyme.

Protein motions and enzyme catalysis are often linked. It is hypothesized that ultrafast vibrations (femtosecond-picosecond) enhance the rate of hydride transfer catalyzed by members of the old yellow enzyme (OYE) family of ene-reductases. Here, we use time-resolved infrared (TRIR) spectroscopy in combination with stable "heavy" isotopic labeling (2H, 13C, 15N) of protein and/or cofactor to probe the vibrational energy transfer (VET) between pentaerythritol tetranitrate reductase (a member of the OYE family) and its noncovalently bound flavin mononucleotide (FMN) cofactor. We show that when the FMN cofactor is photoexcited with visible light, vibrational energy is transferred from the flavin to the surrounding protein environment on the picosecond timescale. This finding expands the scope of VET investigation in proteins, which are limited by suitable intrinsic probes, and may have implications in the understanding of the mechanism of recently discovered photoactive flavoenzymes.

Unravelling the Mechanism of Excited-State Interligand Energy Transfer and the Engineering of Dual Emission in [Ir(C∧N)2(N∧N)]+ Complexes.

Fundamental insights into the mechanism of triplet-excited-state interligand energy transfer dynamics and the origin of dual emission for phosphorescent iridium(III) complexes are presented. The complexes [Ir(C∧N)2(N∧N)]+ (HC∧N = 2-phenylpyridine (1a-c), 2-(2,4-difluorophenyl)pyridine (2a-c), 1-benzyl-4-phenyl-1,2,3-triazole (3a-c); N∧N = 1-benzyl-4-(pyrid-2-yl)-1,2,3-triazole (pytz, a), 1-benzyl-4-(pyrimidin-2-yl)-1,2,3-triazole (pymtz, b), 1-benzyl-4-(pyrazin-2-yl)-1,2,3-triazole (pyztz, c)) are phosphorescent in room-temperature fluid solutions from triplet metal-to-ligand charge transfer (3MLCT) states admixed with either ligand-centered (3LC) (1a, 2a, and 2b) or ligand-to-ligand charge transfer (3LL'CT) character (1c, 2c, and 3a-c). Particularly striking is the observation that pyrimidine-based complex 1b exhibits dual emission from both 3MLCT/3LC and 3MLCT/3LL'CT states. At 77 K, the 3MLCT/3LL'CT component is lost from the photoluminescence spectra of 1b, with emission exclusively arising from its 3MLCT/3LC state, while for 2c switching from 3MLCT/3LL'CT- to 3MLCT/3LC-based emission is observed. Femtosecond transient absorption data reveal distinct spectral signatures characteristic of the population of 3MLCT/3LC states for 1a, 2a, and 2b which persist throughout the 3 ns time frame of the experiment. These 3MLCT/3LC state signatures are apparent in the transient absorption spectra for 1c and 2c immediately following photoexcitation but rapidly evolve to yield spectral profiles characteristic of their 3MLCT/3LL'CT states. Transient data for 1b reveals intermediate behavior: the spectral features of the initially populated 3MLCT/3LC state also undergo rapid evolution, although to a lesser extent than that observed for 1c and 2c, behavior assigned to the equilibration of the 3MLCT/3LC and 3MLCT/3LL'CT states. Density functional theory (DFT) calculations enabled minima to be optimized for both 3MLCT/3LC and 3MLCT/3LL'CT states of 1a-c and 2a-c. Indeed, two distinct 3MLCT/3LC minima were optimized for 1a, 1b, 2a, and 2b distinguished by upon which of the two C∧N ligands the excited electron resides. The 3MLCT/3LC and 3MLCT/3LL'CT states for 1b are very close in energy, in excellent agreement with experimental data demonstrating dual emission. Calculated vibrationally resolved emission spectra (VRES) for the complexes are in excellent agreement with experimental data, with the overlay of spectral maxima arising from emission from the 3MLCT/3LC and 3MLCT/3LL'CT states of 1b convincingly reproducing the observed experimental spectral features. Analysis of the optimized excited-state geometries enable the key structural differences between the 3MLCT/3LC and 3MLCT/3LL'CT states of the complexes to be identified and quantified. The calculation of interconversion pathways between triplet excited states provides for the first time a through-space mechanism for a photoinduced interligand energy transfer process. Furthermore, examination of structural changes between the possible emitting triplet excited states reveals the key bond vibrations that mediate energy transfer between these states. This work therefore provides for the first time detailed mechanistic insights into the fundamental photophysical processes of this important class of complexes.

Observation of the Δg mechanism resulting from the ultrafast spin dynamics that follow the photolysis of coenzyme B12.

Throughout nature, both free radicals and transient radical reaction intermediates are vital to many biological functions. Coenzyme B12 is a case in point. This organometallic cofactor generates a radical pair upon activation in its dependent enzymes by substrate binding and following photolysis. The resulting cob(ii)alamin/5'-deoxyadenosyl radical pair has unusual magnetic properties that present a challenge to detailed investigation at ambient temperatures. Here, we use femtosecond transient absorption spectroscopy adapted for magnetic field exposure to reveal that the spin dynamics of the B12 radical pair are sufficiently fast for magnetic field effects to be observed on the ultrafast reaction kinetics. Moreover, the large difference in g-values between the radicals of the pair means that effects of the Δg mechanism are observed for the first time for a radical pair system exposed to magnetic fields below 1 T. Spin dynamic simulations allow a value of the cob(ii)alamin radical g-value (2.105) at ambient temperature to be extracted and, because the spin dynamic time scale is faster than the diffusional rotation of the cob(ii)alamin radical, the observed value corresponds to the anisotropic g|| value for this radical.

Structural basis for enzymatic photocatalysis in chlorophyll biosynthesis.

The enzyme protochlorophyllide oxidoreductase (POR) catalyses a light-dependent step in chlorophyll biosynthesis that is essential to photosynthesis and, ultimately, all life on Earth1-3. POR, which is one of three known light-dependent enzymes4,5, catalyses reduction of the photosensitizer and substrate protochlorophyllide to form the pigment chlorophyllide. Despite its biological importance, the structural basis for POR photocatalysis has remained unknown. Here we report crystal structures of cyanobacterial PORs from Thermosynechococcus elongatus and Synechocystis sp. in their free forms, and in complex with the nicotinamide coenzyme. Our structural models and simulations of the ternary protochlorophyllide-NADPH-POR complex identify multiple interactions in the POR active site that are important for protochlorophyllide binding, photosensitization and photochemical conversion to chlorophyllide. We demonstrate the importance of active-site architecture and protochlorophyllide structure in driving POR photochemistry in experiments using POR variants and protochlorophyllide analogues. These studies reveal how the POR active site facilitates light-driven reduction of protochlorophyllide by localized hydride transfer from NADPH and long-range proton transfer along structurally defined proton-transfer pathways.

Photochemical Spin Dynamics of the Vitamin B12 Derivative, Methylcobalamin.

Derivatives of vitamin B12 are six-coordinate cobalt corrinoids found in humans, other animals, and microorganisms. By acting as enzymatic cofactors and photoreceptor chromophores, they serve vital metabolic and photoprotective functions. Depending on the context, the chemical mechanisms of the biologically active derivatives of B12-methylcobalamin (MeCbl) and 5'-deoxyadenosylcobalamin (AdoCbl)-can be very different from one another. The extent to which this chemistry is tuned by the upper axial ligand, however, is not yet clear. Here, we have used a combination of time-resolved Fourier transform-electron paramagnetic resonance (FT-EPR), magnetic field effect experiments, and spin dynamic simulations to reveal that the upper axial ligand alone only results in relatively minor changes to the photochemical spin dynamics of B12. By studying the photolysis of MeCbl, we find that, similar to AdoCbl, the initial (or "geminate") radical pairs (RPs) are born predominantly in the singlet spin state and thus originate from singlet excited-state precursors. This is in contrast to the triplet RPs and precursors proposed previously. Unlike AdoCbl, the extent of geminate recombination is limited following MeCbl photolysis, resulting in significant distortions to the FT-EPR signal caused by polarization from spin-correlated methyl-methyl radical "f-pairs" formed following rapid diffusion. Despite the photophysical mechanism that precedes photolysis of MeCbl showing wavelength dependence, the subsequent spin dynamics appear to be largely independent of excitation wavelength, again similar to AdoCbl. Our data finally provide clarity to what in the literature to date has been a confused and contradictory picture. We conclude that, although the upper axial position of MeCbl and AdoCbl does impact their reactivity to some extent, the remarkable biochemical diversity of these fascinating molecules is most likely a result of tuning by their protein environment.

Light-induced structural changes in a full-length cyanobacterial phytochrome probed by time-resolved X-ray scattering.

Phytochromes are photoreceptor proteins that transmit a light signal from a photosensory region to an output domain. Photoconversion involves protein conformational changes whose nature is not fully understood. Here, we use time-resolved X-ray scattering and optical spectroscopy to study the kinetics of structural changes in a full-length cyanobacterial phytochrome and in a truncated form with no output domain. X-ray and spectroscopic signals on the µs/ms timescale are largely independent of the presence of the output domain. On longer time-scales, large differences between the full-length and truncated proteins indicate the timeframe during which the structural transition is transmitted from the photosensory region to the output domain and represent a large quaternary motion. The suggested independence of the photosensory-region dynamics on the µs/ms timescale defines a time window in which the photoreaction can be characterized (e.g. for optogenetic design) independently of the nature of the engineered output domain.

Confinement Effects and Charge Dynamics in Zn3N2 Colloidal Quantum Dots: Implications for QD-LED Displays.

Zinc nitride (Zn3N2) colloidal quantum dots are composed of nontoxic, low-cost, and earth-abundant elements. The effects of quantum confinement on the optical properties and charge dynamics of these dots are studied using steady-state optical characterization and ultrafast fluence-dependent transient absorption. The absorption and emission energies are observed to be size-tunable, with the optical band gap increasing from 1.5 to 3.2 eV as the dot diameter decreased from 8.9 to 2.7 nm. Size-dependent absorption cross sections (σ = 1.22 ± 0.02 × 10-15 to 2.04 ± 0.03 × 10-15 cm2), single exciton lifetimes (0.36 ± 0.02 to 0.65 ± 0.03 ns), as well as Auger recombination lifetimes of biexcitons (3.2 ± 0.4 to 5.0 ± 0.1 ps) and trions (20.8 ± 1.8 to 46.3 ± 1.3 ps) are also measured. The degeneracy of the conduction band minimum (g = 2) is determined from the analysis of the transient absorption spectra at different excitation fluences. The performance of Zn3N2 colloidal quantum dots thus broadly matches that of established visible light emitting quantum dots based on toxic or rare elements, making them a viable alternative for QD-LED displays.

Photochemical Mechanism of an Atypical Algal Phytochrome.

Phytochromes are bilin-containing photoreceptors that are typically sensitive to the red/far-red region of the visible spectrum. Recently, phytochromes from certain eukaryotic algae have become attractive targets for optogenetic applications because of their unique ability to respond to multiple wavelengths of light. Herein, a combination of time-resolved spectroscopy and structural approaches across picosecond to second timescales have been used to map photochemical mechanisms and structural changes in this atypical group of phytochromes. The photochemistry of an orange/far-red light-sensitive algal phytochrome from Dolihomastix tenuilepis has been investigated by using a combination of visible, IR and X-ray scattering probes. The entire photocycle, correlated with accompanying structural changes in the cofactor/protein, are reported. This study identifies a complex photocycle for this atypical phytochrome. It also highlights a need to combine outcomes from a range of biophysical approaches to unravel complex photochemical and macromolecular processes in multi-domain photoreceptor proteins that are the basis of biological light-mediated signalling.

The passivating effect of cadmium in PbS/CdS colloidal quantum dots probed by nm-scale depth profiling.

Achieving control of the surface chemistry of colloidal quantum dots (CQDs) is essential to fully exploit their properties in solar cells, but direct measurement of the chemistry and electronic structure in the outermost atomic layers is challenging. Here we probe the surface oxidation and passivation of cation-exchanged PbS/CdS core/shell CQDs with sub nm-scale precision using synchrotron-radiation-excited depth-profiling photoemission. We investigate the surface composition of the topmost 1-2.5 nm of the CQDs as a function of depth, for CQDs of varying CdS shell thickness, and examine how the surface changes after prolonged air exposure. We demonstrate that the Cd is localized at the surface of the CQDs. The surface-localized products of oxidation are identified, and the extent of oxidation quantified. We show that oxidised sulfur species are progressively eliminated as Cd replaces Pb at the surface. A sub-monolayer surface 'decoration' of Cd is found to be effective in passivating the CQDs. We show that the measured energy-level alignments at PbS/CdS colloidal quantum dot surfaces differ from those expected on the basis of bulk band offsets, and are strongly affected by the oxidation products. We develop a model for the passivating action of Cd. The optimum shell thickness (of around 0.1 nm, previously found to give maximised power conversion efficiency in PbS/CdS solar cells) is found to correspond to a trade-off between the rate of oxidation and the introduction of a surface barrier to charge transport.