Christine Kirmaier

In all photosynthetic reaction centers (RC), transmembrane electron-transfer (ET) cofactor pathways are coordinated by homologous peptides and arranged with approximate C ₂ symmetry, providing two possible paths for charge separation (designated A and B). In type II RCs—Photosystem II and the purple bacterial RCs—only the A branch is active for ET. A variant bacterial RC containing nine amino acid substitutions that result in high-yield ET along the normally nonfunctional B branch was designed. Structural and theoretical studies were combined to understand factors that control unique ET reactions in the complex. Serial femtosecond crystallography performed at an X-ray Free Electron Laser afforded the room temperature X-ray-damage-free crystal structure of the variant RC, revealing interactions between the substituted amino acids and the ET cofactors. The positions of the bacteriochlorin cofactors on the symmetry-related pathways are unaffected, underscoring that the substitutions alter the energetics of ET, leading to the functional changes. In the binding pockets for the terminal electron acceptor quinones, alterations in the positions of neighboring amino acids and water molecules are coordinated with the change of the substituted side chains. Modifications deactivated A-branch ET and activated transmembrane charge separation along the B branch. Electrostatic calculations based on the structure reveal how differences in stabilization of charge-separated states by the protein environment underlie the change in direction of electron flow in this variant RC and provide insights into mechanisms by which B-branch charge separation across the photosynthetic membrane can be achieved in high yield.

Photosynthetic reaction centers carry out a series of electron transfer reactions that convert the light energy into reduced products with chemical energy. However, only the A branch is active despite the structural symmetry.

Photosynthetic reaction centers carry out a series of electron and proton transfer reactions that convert the energy of light into high energy reduced products and add to the proton gradient. All photosynthetic reaction centers (Photosystems I, II, Heliobacter, and bacterial reaction centers (bRCs)) have their charge separating cofactors arranged with two, c2-symmetric paths from primary donor to terminal acceptor. In Type II reaction centers (PSII and bRCs), electrons utilize only one active branch. In bRCs the electron transfer occurs in the A branch from the excited state of dimer P860 through BChlA, BPhA, to QA, and then to the B-branch QB. B-branch BChlB and BPhB do not participate. A similar path is found in PSII. These proteins challenge our understanding of how proteins can turn on or off the activity of bound ligands. The shift of the electrochemical midpoint potential (Em) of each cofactor by the protein environment is calculated using the continuum electrostatics within the MultiConformation Continuum Electrostatic program (MCCE). In bRCs from Rhodobacter sphaeroides, the electrostatic potentials favor reduction of the active branch cofactors. The residues that contribute to a more positive potential on the A branch are identified. These are often distant from the cofactors. The large difference of the potential at the competing BChls may direct the electron transfer to the A branch. Residues TyrM210 and PheL181 are at symmetrical positions on the A and B branches, near P860 and between BChlA or BChlB. Mutated bRCs that swap the Tyr and Phe (YF mutant), have been shown to support some electron transfer to the B-branch cofactors. The calculated Ems in the bRC with the YF swap show that more positive B-branch potentials help explain the electron transfer from excited P860 to the normally inactive B branch.

Intraprotein electron transfer (ET) requires explicit local control of the environment of cofactors to influence their intermolecular distances, relative orientations, and redox properties. Efficient, longer-range ET often utilizes molecular orbitals of aromatic residues present in the intervening space. Here, revitalization of a vestigial ET pathway in the bacterial photosynthetic reaction center is achieved by scanning with tryptophans to uncover markedly improved routes of electron conduction in a key stabilizing step spanning 15 Å between tetrapyrrole and quinone cofactors. This ET event is maximally enhanced by pairing one or more tryptophans with a threonine to influence quinone binding and/or redox potential. Synergistic effects of these substitutions increase the yield of that ET step to ~95%. Joining these substitutions with mutant residues that improve initial ET steps dramatically enhances transmembrane charge separation via this redesigned version of a pathway that is quantitatively inactive in the native protein-cofactor complex.

The initial light-induced electron transfer (ET) steps in the bacterial photosynthetic reaction center (RC) have been extensively studied and provide a paradigm for connecting structure and function. Although RCs have local pseudo-C₂ symmetry, ET only occurs along the A branch of chromophores. Tyrosine M210 is a key symmetry-breaking residue adjacent to bacteriochlorophyll B_A that bridges the primary electron donor P and the bacteriopheophytin acceptor H_A. We used amber suppression to incorporate phenylalanine variants with different electron-withdrawing/-donating capabilities at the position M210. X-ray data generally reveal no appreciable structural changes due to the mutations. P* decay and P⁺H_A^- formation are multiexponential (∼2 to 9, ∼10 to 60, and ∼100 to 300 ps) and temperature dependent. The 1020 nm transient-absorption band of P⁺B_A^- is barely resolved for a few variants at 295 K and for none at 77 K. The results indicate a change from two-step ET for wild-type RCs to the dominance of one-step superexchange ET for the mutants. Resonance Stark spectroscopy reveals that the free energy of P⁺B_A^- changes by -57 to +66 meV among the phenylalanine variants. Because P⁺B_A^- apparently lies above P* in all phenylalanine variants, the perturbations primarily affect the energy denominator for superexchange mixing. The findings deepen insight into primary ET in the bacterial RC.

The initial light-induced electron transfer (ET) steps in the bacterial photosynthetic reaction center (RC) have been extensively studied and provide a paradigm for connecting structure and function. Although RCs have local pseudo-C2 symmetry, ET only occurs along the A branch of chromophores. Tyrosine M210 is a key symmetry-breaking residue adjacent to bacteriochlorophyll BA that bridges primary electron donor P and bacteriopheophytin acceptor HA. We used amber suppression to incorporate phenylalanine variants with different electron-withdrawing/donating capabilities at position M210. X-ray data generally reveal no appreciable structural changes due to the mutations. P* decay and P+HA formation are multi-exponential (~2-9, ~10-60, and ~100-300 ps) and temperature dependent. The 1020 nm transient-absorption band of P+BA is barely resolved for a few variants at 295 K and for none at 77 K. The results indicate a change from two-step ET for wild-type RCs to dominance of one-step superexchange ET for the mutants. Resonance Stark spectroscopy reveals that the free energy of P+BA changes by -57 to +66 meV among the phenylalanine variants. Because P+BA apparently lies above P* in all phenylalanine variants, the perturbations primarily affect the energy denominator for superexchange mixing. The findings deepen insight into primary ET in the bacterial RC.

The ability to capture and utilize light in the near-ultraviolet (NUV), visible and near-infrared (NIR-I and NIR-II) spectral regions (i.e., 320–400, 400–700, 700–1000, 10001700 nm) is essential for any solar-energy conversion scheme. Nature employs chlorophylls and bacteriochlorophylls in light-harvesting architectures to absorb light in the blue and red/NIR regions. Accessory pigments (carotenoids, bilins) augment absorption of the (bacterio)chlorophylls in the green region. The harvested energy is funneled to a reaction center protein, where charge separation occurs. Subsequent migration of the electron and the hole stabilizes and stores the energy from light via redox chemistry. The long-term objective of the Bocian/Holten&Kirmaier/Lindsey research program under this DOE grant has been to develop tetrapyrrole-based molecular architectures that absorb sunlight, funnel energy and separate charge with high efficiency. Integral to the program has been iterative cycles of design, synthesis and characterization that provided deep insights into the relationships between chemical composition, electronic structure, and key static and dynamic properties (vibrational, redox, photophysical, energy/charge transfer) of tetrapyrrolic systems. Such architectures included monomers, dyads, larger arrays, and complexes with accessory components. The objective was to develop molecular designs and guiding principles to enhance current and future energy-conversion schemes. Molecular arrays targeted to address one or more fundamental questions concerning light harvesting and energy/charge transfer were constructed from analogues of the naturally occurring hemes, chlorophylls and bacteriochlorophylls. Diverse, tunable synthetic building blocks were prepared that spanned the three respective tetrapyrrole families, which are the porphyrins, chlorins and bacteriochlorins. Thus, the research focused on porphyrins as well as synthetic surrogates for chlorophylls (chlorins, 131-oxophorbines and chlorin-imides) and bacteriochlorophylls (bacteriochlorins, bacterio-131-oxophorbines and bacteriochlorin-imides), generically termed hydroporphyrins. Although the three tetrapyrrole classes (porphyrins, chlorins and bacteriochlorins) absorb light strongly in the violet-blue spectral region, the long-wavelength absorption band typically lies in the green-orange, red, and NIR regions, respectively, with increasing intensity. Understanding the spectra, electronic structure, and energy/charge-transfer properties of such tetrapyrrolic macrocycles is of central importance for the rational design of molecular architectures for solar-energy conversion. Our integrated program of molecular design and synthesis coupled with a variety of spectroscopic, electrochemical, and computational studies have probed from first principles how structural and electronic properties of tetrapyrrolic macrocycles dictate spectral properties as well as the rates of ground-state hole/electron transfer and excited-state energy flow in multicomponent architectures. Individual molecules and multicomponent architectures were designed to test ideas of fundamental importance, often requiring the development of new synthetic methodology. The members of the collaborative team had almost daily discussions by phone and/or e-mail concerning design of molecules, flow of compounds between the labs, planning of physical characterization studies, discussing results and analysis and integrating into design of next generation architectures, and the preparation of manuscripts. Furthermore, students and postdocs in the different labs routinely communicated with one another to facilitate the advancement of the research activities. In short, a highly integrated and collaborative research program was well established among the groups. The research effort involved molecular design and synthesis of synthetic molecular architectures by the Lindsey group integrated with physicochemical and photophysical characterization by the Bocian group and the Holten&Kirmaier group (Figure 2). The Bocian group carried out electrochemical, electron paramagnetic resonance (EPR), resonance Raman (RR), and Fourier-transform infrared (FT-IR) studies, as well as density functional theory (DFT) calculations and the time-dependent extension (TDDFT) to gain insight into excited-state properties. The Holten&Kirmaier group carried out static and time-resolved absorption and fluorescence spectroscopy studies and simulated absorption spectra using molecular orbital (MO) energies from DFT as input to the four-orbital model to complement the TDDFT calculations. The combined measurements provided understanding of the vibrational/electronic properties of the individual molecules and the changes that occur upon incorporation into multicomponent architectures. This information underpinned elucidating the mechanisms and timescales of ground-state hole/electron transfer and excited-state energy and charge transfer.

Two annulated bacteriochlorin isomers exhibit profoundly different spectral and photophysical features, which are attributed to the absence or presence of electronic (and vibronic) mixing of x - and y -polarized transitions/states.

Bacteriochlorophylls, nature's near-infrared absorbers, play an essential role in energy transfer in photosynthetic antennas and reaction centers. To probe energy-transfer processes akin to those in photosynthetic systems, nine synthetic bacteriochlorin–bacteriochlorin dyads have been prepared wherein the constituent pigments are joined at the meso-positions by a phenylethyne linker. The phenylethyne linker is an unsymmetric auxochrome, which differentially shifts the excited-state energies of the phenyl- or ethynyl-attached bacteriochlorin constituents in the dyad. Molecular designs utilized known effects of macrocycle substituents to engineer bacteriochlorins with S₀ → S₁ (Q_y) transitions spanning 725–788 nm. The design-predicted donor–acceptor excited-state energy gaps in the dyads agree well with those obtained from time dependent density functional theory calculations and with the measured range of 197–1089 cm⁻¹. Similar trends with donor–acceptor excited-state energy gaps are found for (1) the measured ultrafast energy-transfer rates of (0.3–1.7 ps)⁻¹, (2) the spectral overlap integral (J) in Förster energy-transfer theory, and (3) donor–acceptor electronic mixing manifested in the natural transition orbitals for the S₀ → S₁ transition. Subtle outcomes include the near orthogonal orientation of the π-planes of the bacteriochlorin macrocycles, and the substituent-induced shift in transition-dipole moment from the typical coincidence with the NH–NH axis; the two features together afforded the Förster orientation term κ² ranging from 0.55–1.53 across the nine dyads, a value supportive of efficient excited-state energy transfer. The molecular design and collective insights on the dyads are valuable for studies relevant to artificial photosynthesis and other processes requiring ultrafast energy transfer.