Photosynthetic reaction center variants made via genetic code expansion show Tyr at M210 tunes the initial electron transfer mechanism.

Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides were engineered to vary the electronic properties of a key tyrosine (M210) close to an essential electron transfer component via its replacement with site-specific, genetically encoded noncanonical amino acid tyrosine analogs. High fidelity of noncanonical amino acid incorporation was verified with mass spectrometry and X-ray crystallography and demonstrated that RC variants exhibit no significant structural alterations relative to wild type (WT). Ultrafast transient absorption spectroscopy indicates the excited primary electron donor, P*, decays via a ∼4-ps and a ∼20-ps population to produce the charge-separated state P+HA- in all variants. Global analysis indicates that in the ∼4-ps population, P+HA- forms through a two-step process, P*→ P+BA-→ P+HA-, while in the ∼20-ps population, it forms via a one-step P* → P+HA- superexchange mechanism. The percentage of the P* population that decays via the superexchange route varies from ∼25 to ∼45% among variants, while in WT, this percentage is ∼15%. Increases in the P* population that decays via superexchange correlate with increases in the free energy of the P+BA- intermediate caused by a given M210 tyrosine analog. This was experimentally estimated through resonance Stark spectroscopy, redox titrations, and near-infrared absorption measurements. As the most energetically perturbative variant, 3-nitrotyrosine at M210 creates an ∼110-meV increase in the free energy of P+BA- along with a dramatic diminution of the 1,030-nm transient absorption band indicative of P+BA- formation. Collectively, this work indicates the tyrosine at M210 tunes the mechanism of primary electron transfer in the RC.

Switching sides-Reengineered primary charge separation in the bacterial photosynthetic reaction center.

We report 90% yield of electron transfer (ET) from the singlet excited state P* of the primary electron-donor P (a bacteriochlorophyll dimer) to the B-side bacteriopheophytin (HB) in the bacterial photosynthetic reaction center (RC). Starting from a platform Rhodobacter sphaeroides RC bearing several amino acid changes, an Arg in place of the native Leu at L185-positioned over one face of HB and only ∼4 Šfrom the 4 central nitrogens of the HB macrocycle-is the key additional mutation providing 90% yield of P+HB- This all but matches the near-unity yield of A-side P+HA- charge separation in the native RC. The 90% yield of ET to HB derives from (minimally) 3 P* populations with distinct means of P* decay. In an ∼40% population, P* decays in ∼4 ps via a 2-step process involving a short-lived P+BB- intermediate, analogous to initial charge separation on the A side of wild-type RCs. In an ∼50% population, P* → P+HB- conversion takes place in ∼20 ps by a superexchange mechanism mediated by BB An ∼10% population of P* decays in ∼150 ps largely by internal conversion. These results address the long-standing dichotomy of A- versus B-side initial charge separation in native RCs and have implications for the mechanism(s) and timescale of initial ET that are required to achieve a near-quantitative yield of unidirectional charge separation.

In Situ, Protein-Mediated Generation of a Photochemically Active Chlorophyll Analogue in a Mutant Bacterial Photosynthetic Reaction Center.

All possible natural amino acids have been substituted for the native LeuL185 positioned near the B-side bacteriopheophytin (HB) in the bacterial reaction center (RC) from Rhodobacter sphaeroides. Additional mutations that enhance electron transfer to the normally inactive B-side cofactors are present. Approximately half of the isolated RCs with Glu at L185 contain a magnesium chlorin (CB) in place of HB. The chlorin is not the common BChl a oxidation product 3-desvinyl-3-acetyl chlorophyll a with a C-C bond in ring D and a C═C bond in ring B but has properties consistent with reversal of these bond orders, giving 17,18-didehydro BChl a. In such RCs, charge-separated state P+CB- forms in ∼5% yield. The other half of the GluL185-containing RCs have a bacteriochlorophyll a (BChl a) denoted ßB in place of HB. Residues His, Asp, Asn, and Gln at L185 yield RCs with ≥85% ßB in the HB site, while most other amino acids result in RCs that retain HB (≥95%). To the best of our knowledge, neither bacterial RCs that harbor five BChl a molecules and one chlorophyll analogue nor those with six BChl a molecules have been reported previously. The finding that altering the local environment within a cofactor binding site of a transmembrane complex leads to in situ generation of a photoactive chlorin with an unusual ring oxidation pattern suggests new strategies for amino acid control over pigment type at specific sites in photosynthetic proteins.

Consequences of saturation mutagenesis of the protein ligand to the B-side monomeric bacteriochlorophyll in reaction centers from Rhodobacter capsulatus.

In bacterial reaction centers (RCs), photon-induced initial charge separation uses an A-side bacteriochlorophyll (BChl, BA) and bacteriopheophytin (BPh, HA), while the near-mirror image B-side BB and HB cofactors are inactive. Two new sets of Rhodobacter capsulatus RC mutants were designed, both bearing substitution of all amino acids for the native histidine M180 (M-polypeptide residue 180) ligand to the core Mg ion of BB. Residues are identified that largely result in retention of a BChl in the BB site (Asp, Ser, Pro, Gln, Asn, Gly, Cys, Lys, and Thr), ones that largely harbor the Mg-free BPh in the BB site (Leu and Ile), and ones for which isolated RCs are comprised of a substantial mixture of these two RC types (Ala, Glu, Val, Met and, in one set, Arg). No protein was isolated when M180 is Trp, Tyr, Phe, or (in one set) Arg. These findings are corroborated by ground state spectra, pigment extractions, ultrafast transient absorption studies, and the yields of B-side transmembrane charge separation. The changes in coordination chemistries did not reveal an RC with sufficiently precise poising of the redox properties of the BB-site cofactor to result in a high yield of B-side electron transfer to HB. Insights are gleaned into the amino acid properties that support BChl in the BB site and into the widely observed multi-exponential decay of the excited state of the primary electron donor. The results also have direct implications for tuning free energies of the charge-separated intermediates in RCs and mimetic systems.

Optimizing multi-step B-side charge separation in photosynthetic reaction centers from Rhodobacter capsulatus.

Using high-throughput methods for mutagenesis, protein isolation and charge-separation functionality, we have assayed 40 Rhodobacter capsulatus reaction center (RC) mutants for their P(+)QB(-) yield (P is a dimer of bacteriochlorophylls and Q is a ubiquinone) as produced using the normally inactive B-side cofactors BB and HB (where B is a bacteriochlorophyll and H is a bacteriopheophytin). Two sets of mutants explore all possible residues at M131 (M polypeptide, native residue Val near HB) in tandem with either a fixed His or a fixed Asn at L181 (L polypeptide, native residue Phe near BB). A third set of mutants explores all possible residues at L181 with a fixed Glu at M131 that can form a hydrogen bond to HB. For each set of mutants, the results of a rapid millisecond screening assay that probes the yield of P(+)QB(-) are compared among that set and to the other mutants reported here or previously. For a subset of eight mutants, the rate constants and yields of the individual B-side electron transfer processes are determined via transient absorption measurements spanning 100 fs to 50 µs. The resulting ranking of mutants for their yield of P(+)QB(-) from ultrafast experiments is in good agreement with that obtained from the millisecond screening assay, further validating the efficient, high-throughput screen for B-side transmembrane charge separation. Results from mutants that individually show progress toward optimization of P(+)HB(-)→P(+)QB(-) electron transfer or initial P*→P(+)HB(-) conversion highlight unmet challenges of optimizing both processes simultaneously.

High yield of secondary B-side electron transfer in mutant Rhodobacter capsulatus reaction centers.

From the crystal structures of reaction centers (RCs) from purple photosynthetic bacteria, two pathways for electron transfer (ET) are apparent but only one pathway (the A side) operates in the native protein-cofactor complex. Partial activation of the B-side pathway has unveiled the true inefficiencies of ET processes on that side in comparison to analogous reactions on the A side. Of significance are the relative rate constants for forward ET and the competing charge recombination reactions. On the B side, these rate constants are nearly equal for the secondary charge-separation step (ET from bacteriopheophytin to quinone), relegating the yield of this process to <50%. Herein we report efforts to optimize this step. In surveying all possible residues at position 131 in the M subunit, we discovered that when glutamic acid replaces the native valine the efficiency of the secondary ET is nearly two-fold higher than in the wild-type RC. The positive effect of M131 Glu is likely due to formation of a hydrogen bond with the ring V keto group of the B-side bacteriopheophytin leading to stabilization of the charge-separated state involving this cofactor. This change slows charge recombination by roughly a factor of two and affords the improved yield of the desired forward ET to the B-side quinone terminal acceptor.

Two pathways to understanding electron transfer in reaction centers from photosynthetic bacteria: A comparison of Rhodobacter sphaeroides and Rhodobacter capsulatus mutants.

The rates, yields, mechanisms and directionality of electron transfer (ET) are explored in twelve pairs of Rhodobacter (R.) sphaeroides and R. capsulatus mutant RCs designed to defeat ET from the excited primary donor (P*) to the A-side cofactors and re-direct ET to the normally inactive mirror-image B-side cofactors. In general, the R. sphaeroides variants have larger P+HB- yields (up to ∼90%) than their R. capsulatus analogs (up to ∼60%), where HB is the B-side bacteriopheophytin. Substitution of Tyr for Phe at L-polypeptide position L181 near BB primarily increases the contribution of fast P* â†’ P+BB- â†’ P+HB- two-step ET, where BB is the "bridging" B-side bacteriochlorophyll. The second step (∼6-8 ps) is slower than the first (∼3-4 ps), unlike A-side two-step ET (P* â†’ P+BA- â†’ P+HA-) where the second step (∼1 ps) is faster than the first (∼3-4 ps) in the native RC. Substitutions near HB, at L185 (Leu, Trp or Arg) and at M-polypeptide site M133/131 (Thr, Val or Glu), strongly affect the contribution of slower (20-50 ps) P* â†’ P+HB- one-step superexchange ET. Both ET mechanisms are effective in directing electrons "the wrong way" to HB and both compete with internal conversion of P* to the ground state (∼200 ps) and ET to the A-side cofactors. Collectively, the work demonstrates cooperative amino-acid control of rates, yields and mechanisms of ET in bacterial RCs and how A- vs. B-side charge separation can be tuned in both species.

High throughput engineering to revitalize a vestigial electron transfer pathway in bacterial photosynthetic reaction centers.

Photosynthetic reaction centers convert light energy into chemical energy in a series of transmembrane electron transfer reactions, each with near 100% yield. The structures of reaction centers reveal two symmetry-related branches of cofactors (denoted A and B) that are functionally asymmetric; purple bacterial reaction centers use the A pathway exclusively. Previously, site-specific mutagenesis has yielded reaction centers capable of transmembrane charge separation solely via the B branch cofactors, but the best overall electron transfer yields are still low. In an attempt to better realize the architectural and energetic factors that underlie the directionality and yields of electron transfer, sites within the protein-cofactor complex were targeted in a directed molecular evolution strategy that implements streamlined mutagenesis and high throughput spectroscopic screening. The polycistronic approach enables efficient construction and expression of a large number of variants of a heteroligomeric complex that has two intimately regulated subunits with high sequence similarity, common features of many prokaryotic and eukaryotic transmembrane protein assemblies. The strategy has succeeded in the discovery of several mutant reaction centers with increased efficiency of the B pathway; they carry multiple substitutions that have not been explored or linked using traditional approaches. This work expands our understanding of the structure-function relationships that dictate the efficiency of biological energy-conversion reactions, concepts that will aid the design of bio-inspired assemblies capable of both efficient charge separation and charge stabilization.

High Yield of B-Side Electron Transfer at 77 K in the Photosynthetic Reaction Center Protein from Rhodobacter sphaeroides.

The primary electron transfer (ET) processes at 295 and 77 K are compared for the Rhodobacter sphaeroides reaction center (RC) pigment-protein complex from 13 mutants including a wild-type control. The engineered RCs bear mutations in the L and M polypeptides that largely inhibit ET from the excited state P* of the primary electron donor (P, a bacteriochlorophyll dimer) to the normally photoactive A-side cofactors and enhance ET to the C2-symmetry related, and normally photoinactive, B-side cofactors. P* decay is multiexponential at both temperatures and modeled as arising from subpopulations that differ in contributions of two-step ET (e.g., P* → P+BB- → P+HB-), one-step superexchange ET (e.g., P* → P+HB-), and P* → ground state. [HB and BB are monomeric bacteriopheophytin and bacteriochlorophyll, respectively.] The relative abundances of the subpopulations and the inherent rate constants of the P* decay routes vary with temperature. Regardless, ET to produce P+HB- is generally faster at 77 K than at 295 K by about a factor of 2. A key finding is that the yield of P+HB-, which ranges from ∼5% to ∼90% among the mutant RCs, is essentially the same at 77 K as at 295 K in each case. Overall, the results show that ET from P* to the B-side cofactors in these mutants does not require thermal activation and involves combinations of ET mechanisms analogous to those operative on the A side in the native RC.

Structures of Rhodopseudomonas palustris RC-LH1 complexes with open or closed quinone channels.

The reaction-center light-harvesting complex 1 (RC-LH1) is the core photosynthetic component in purple phototrophic bacteria. We present two cryo-electron microscopy structures of RC-LH1 complexes from Rhodopseudomonas palustris A 2.65-Å resolution structure of the RC-LH114-W complex consists of an open 14-subunit LH1 ring surrounding the RC interrupted by protein-W, whereas the complex without protein-W at 2.80-Å resolution comprises an RC completely encircled by a closed, 16-subunit LH1 ring. Comparison of these structures provides insights into quinone dynamics within RC-LH1 complexes, including a previously unidentified conformational change upon quinone binding at the RC QB site, and the locations of accessory quinone binding sites that aid their delivery to the RC. The structurally unique protein-W prevents LH1 ring closure, creating a channel for accelerated quinone/quinol exchange.

Engineering of B800 bacteriochlorophyll binding site specificity in the Rhodobacter sphaeroides LH2 antenna.

The light-harvesting 2 complex (LH2) of the purple phototrophic bacterium Rhodobacter sphaeroides is a highly efficient, light-harvesting antenna that allows growth under a wide-range of light intensities. In order to expand the spectral range of this antenna complex, we first used a series of competition assays to measure the capacity of the non-native pigments 3-acetyl chlorophyll (Chl) a, Chl d, Chl f or bacteriochlorophyll (BChl) b to replace native BChl a in the B800 binding site of LH2. We then adjusted the B800 site and systematically assessed the binding of non-native pigments. We find that Arg-10 of the LH2 ß polypeptide plays a crucial role in binding specificity, by providing a hydrogen-bond to the 3-acetyl group of native and non-native pigments. Reconstituted LH2 complexes harbouring the series of (B)Chls were examined by transient absorption and steady-state fluorescence spectroscopies. Although slowed 10-fold to ~6 ps, energy transfer from Chl a to B850 BChl a remained highly efficient. We measured faster energy-transfer time constants for Chl d (3.5 ps) and Chl f (2.7 ps), which have red-shifted absorption maxima compared to Chl a. BChl b, red-shifted from the native BChl a, gave extremely rapid (≤0.1 ps) transfer. These results show that modified LH2 complexes, combined with engineered (B)Chl biosynthesis pathways in vivo, have potential for retaining high efficiency whilst acquiring increased spectral range.

Manipulating the Energetics and Rates of Electron Transfer in Rhodobacter capsulatus Reaction Centers with Asymmetric Pigment Content.

Seemingly redundant parallel pathways for electron transfer (ET), composed of identical sets of cofactors, are a cornerstone feature of photosynthetic reaction centers (RCs) involved in light-energy conversion. In native bacterial RCs, both A and B branches house one bacteriochlorophyll (BChl) and one bacteriopheophytin (BPh), but the A branch is used exclusively. Described herein are the results obtained for two Rhodobacter capsulatus RCs with an unnaturally high degree of cofactor asymmetry, two BPh on the RC's B side and two BChl on the A side. These pigment changes derive, respectively, from the His(M180)Leu mutation [a BPh (ΦB) replaces the B-side BChl (BB)], and the Leu(M212)His mutation [a BChl (ßA) replaces the A-side BPh (HA)]. Additionally, Tyr(M208)Phe was employed to disfavor ET to the A branch; in one mutant, Val(M131)Glu creates a hydrogen bond to HB to enhance ET to HB. In both ΦB mutants, the decay kinetics of the excited primary ET donor (P*) resolve three populations with lifetimes of ∼9 ps (50-60%), ∼40 ps (10-20%), and ∼200 ps (20-30%), with P+ΦB- formed predominantly from the 9 ps fraction. The 50-60% yield of P+ΦB- is the highest yet observed for a ΦB-containing RC. The results provide insight into factors needed for efficient multistep ET.

Augmenting light coverage for photosynthesis through YFP-enhanced charge separation at the Rhodobacter sphaeroides reaction centre.

Photosynthesis uses a limited range of the solar spectrum, so enhancing spectral coverage could improve the efficiency of light capture. Here, we show that a hybrid reaction centre (RC)/yellow fluorescent protein (YFP) complex accelerates photosynthetic growth in the bacterium Rhodobacter sphaeroides. The structure of the RC/YFP-light-harvesting 1 (LH1) complex shows the position of YFP attachment to the RC-H subunit, on the cytoplasmic side of the RC complex. Fluorescence lifetime microscopy of whole cells and ultrafast transient absorption spectroscopy of purified RC/YFP complexes show that the YFP-RC intermolecular distance and spectral overlap between the emission of YFP and the visible-region (QX) absorption bands of the RC allow energy transfer via a Förster mechanism, with an efficiency of 40±10%. This proof-of-principle study demonstrates the feasibility of increasing spectral coverage for harvesting light using non-native genetically-encoded light-absorbers, thereby augmenting energy transfer and trapping in photosynthesis.

Species differences in unlocking B-side electron transfer in bacterial reaction centers.

The structure of the bacterial photosynthetic reaction center (RC) reveals symmetry-related electron transfer (ET) pathways, but only one path is used in native RCs. Analogous mutations have been made in two Rhodobacter (R.) species. A glutamic acid at position 133 in the M subunit increases transmembrane charge separation via the naturally inactive (B-side) path through impacts on primary ET in mutant R. sphaeroidesRCs. Prior work showed that the analogous substitution in the R. capsulatusRC also increases B-side activity, but mainly affects secondary ET. The overall yields of transmembrane ET are similar, but enabled in fundamentally different ways.

Cytogenetic and Sequence Analyses of Mitochondrial DNA Insertions in Nuclear Chromosomes of Maize.

The transfer of mitochondrial DNA (mtDNA) into nuclear genomes is a regularly occurring process that has been observed in many species. Few studies, however, have focused on the variation of nuclear-mtDNA sequences (NUMTs) within a species. This study examined mtDNA insertions within chromosomes of a diverse set of Zea mays ssp. mays (maize) inbred lines by the use of fluorescence in situ hybridization. A relatively large NUMT on the long arm of chromosome 9 (9L) was identified at approximately the same position in four inbred lines (B73, M825, HP301, and Oh7B). Further examination of the similarly positioned 9L NUMT in two lines, B73 and M825, indicated that the large size of these sites is due to the presence of a majority of the mitochondrial genome; however, only portions of this NUMT (~252 kb total) were found in the publically available B73 nuclear sequence for chromosome 9. Fiber-fluorescence in situ hybridization analysis estimated the size of the B73 9L NUMT to be ~1.8 Mb and revealed that the NUMT is methylated. Two regions of mtDNA (2.4 kb and 3.3 kb) within the 9L NUMT are not present in the B73 mitochondrial NB genome; however, these 2.4-kb and 3.3-kb segments are present in other Zea mitochondrial genomes, including that of Zea mays ssp. parviglumis, a progenitor of domesticated maize.

Photophysical Properties and Electronic Structure of Chlorin-Imides: Bridging the Gap between Chlorins and Bacteriochlorins.

Efficient light harvesting for molecular-based solar-conversion systems requires absorbers that span the photon-rich red and near-infrared (NIR) regions of the solar spectrum. Reported herein are the photophysical properties of a set of six chlorin-imides and nine synthetic chlorin analogues that extend the absorption deeper (624-714 nm) into these key spectral regions. These absorbers help bridge the gap between typical chlorins and bacteriochlorins. The new compounds have high fluorescence quantum yields (0.15-0.34) and long singlet excited-state lifetimes (4.2-10.9 ns). The bathochromic shift in Qy absorption is driven by substituent-based stabilization of the lowest unoccupied molecular orbital, with the largest shifts for chlorins that bear an electron-withdrawing, conjugative group at the 3-position in combination with a 13,15-imide ring.

Protein influence on charge-asymmetry of the primary donor in photosynthetic bacterial reaction centers containing a heterodimer: effects on photophysical properties and electron transfer.

The substantial electronic distinctions between bacteriochlorophyll (BChl) and its Mg-free analogue bacteriopheophytin (BPh) are exploited in two sets of Rhodobacter capsulatus reaction center (RC) mutants that contain a heterodimeric BChl-BPh primary electron donor (D). The BPh component of the M-heterodimer (Mhd) or L-heterodimer (Lhd) obtains from substituting a Leu for His M200 or for His L173, respectively. Lhd-ß and Mhd-ß RCs serve as the initial templates in the two mutant sets, where ß denotes that the L-side BPh acceptor (HL) has been replaced by a BChl (due to substituting His for Leu M212). Three variants each of Lhd-ß and Mhd-ß mutants were constructed: (1) a swap (denoted YF) of the native Phe (L181) and Tyr (M208) residues, which flank D and the nearby M- and L-side monomeric BChl cofactors, respectively, giving Tyr (L181) and Phe (M208); (2) addition of a hydrogen bond (denoted L131LH) to the ring V keto group of the L-macrocycle of D, via replacing the native Leu at L131 with His; (3) the combination of 1 and 2. A low yield of electron transfer (ET) to the M-side BPh (HM) is observed in all four Lhd-containing RCs. Comparison with the yield of ET to ß on the L-side shows that electron density on the L-macrocycle of D* favors ET to the M-side cofactors and vice versa. Increasing or decreasing the electronic asymmetry of D* via the YF, L131LH mutations or the combination results in consistent trends in the characteristics of the long-wavelength ground state absorption band of D, the rate constant of internal conversion of D* to the ground state, and the rate constants for ET to both the L- and M-side cofactors. A surprising correlation is that an increase in the charge asymmetry in D* not only increases the D* internal-conversion rate constant, but also the rate constants for ET to both the L- and M-side cofactors, spanning time scales of tens of picoseconds to several nanoseconds. The YF swap has a previously unrecognized effect on the electronic asymmetry of D*, resulting in increased charge asymmetry for the Mhd and decreased charge asymmetry for the Lhd. This result indicates that the native Tyr (M208) and Phe (L181) in the wild-type RC promote an electron distribution in P* that is the reverse of that favorable for ET to the photoactive L-branch. This conclusion reinforces the view that the native configuration of these residues promotes ET to the L branch primarily by poising the free energies of the charge-separated states. Overall, this work addresses the extent to which electronic couplings complement energetics in underpinning the directionality of ET in the bacterial RC.

Effects of substituents on synthetic analogs of chlorophylls. Part 3: The distinctive impact of auxochromes at the 7- versus 3-positions.

Assessing the effects of substituents on the spectra of chlorophylls is essential for gaining a deep understanding of photosynthetic processes. Chlorophyll a and b differ solely in the nature of the 7-substituent (methyl versus formyl), whereas chlorophyll a and d differ solely in the 3-substituent (vinyl versus formyl), yet have distinct long-wavelength absorption maxima: 665 (a) 646 (b) and 692 nm (d). Herein, the spectra, singlet excited-state decay characteristics, and results from DFT calculations are examined for synthetic chlorins and 13(1)-oxophorbines that contain ethynyl, acetyl, formyl and other groups at the 3-, 7- and/or 13-positions. Substituent effects on the absorption spectra are well accounted for using Gouterman's four-orbital model. Key findings are that (1) the dramatic difference in auxochromic effects of a given substituent at the 7- versus 3- or 13-positions primarily derives from relative effects on the LUMO+1 and LUMO; (2) formyl at the 7- or 8-position effectively "porphyrinizes" the chlorin and (3) the substituent effect increases in the order of vinyl < ethynyl < acetyl < formyl. Thus, the spectral properties are governed by an intricate interplay of electronic effects of substituents at particular sites on the four frontier MOs of the chlorin macrocycle.