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 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.

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.

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.

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.

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.

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.

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.

Herpetofaunal community change in multiple habitats after fifteen years in a southwest Florida preserve, USA.

Herpetofaunal declines have been documented globally, and southern Florida, USA, is an especially vulnerable region because of high impacts from hydrological perturbations and nonindigenous species. To assess the extent of recent change in herpetofauna community composition, we established a baseline inventory during 1995-97 at a managed preserve in a habitat rich area of southwest Florida, and repeated our sampling methods fifteen years later (2010-11). Nine drift fence arrays were placed in four habitat types: mesic flatwood, mesic hammock, depression marsh, and wet prairie. Trapping occurred daily for one week during 7-8 sampling runs in each period (57 and 49 total sampling days, respectively). Species richness was maintained in mesic hammock habitats but varied in the others. Catch rates of several native species (Anaxyrus terrestris, Lithobates grylio, Anolis carolinensis, Nerodia fasciata) declined significantly. Other native species (Lithobates sphenocephalus, Siren lacertian, and Notophthalmus viridescens piaropicola) that were abundant in 1995-97 declined by greater than 50%. Catch rate of only two species (the nonindigenous Anolis sagrei and the native Diadophis punctatus) increased significantly. Hierarchical cluster analysis indicated similarity within habitat types but significant dissimilarity between sampling periods, confirming shifts in community composition. Analysis of individual species' contributions to overall similarity across habitats shows a shift from dominance of native species in the 1990s to increased importance of nonindigenous species in 2010-11. Although natural population fluctuations may have influenced differences between the two sampling periods, our results suggest considerable recent change in the structure and composition of this southwest Florida herpetofaunal community. The causes are unknown, but hydrological shifts and ecological impacts of nonindigenous species may have contributed.

Stigmatellin probes the electrostatic potential in the QB site of the photosynthetic reaction center.

The electrostatic potential in the secondary quinone (QB) binding site of the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides determines the rate and free energy change (driving force) of electron transfer to QB. It is controlled by the ionization states of residues in a strongly interacting cluster around the QB site. Reduction of the QB induces change of the ionization states of residues and binding of protons from the bulk. Stigmatellin, an inhibitor of the mitochondrial and photosynthetic respiratory chain, has been proven to be a unique voltage probe of the QB binding pocket. It binds to the QB site with high affinity, and the pK value of its phenolic group monitors the local electrostatic potential with high sensitivity. Investigations with different types of detergent as a model system of isolated RC revealed that the pK of stigmatellin was controlled overwhelmingly by electrostatic and slightly by hydrophobic interactions. Measurements showed a high pK value (>11) of stigmatellin in the QB pocket of the dark-state wild-type RC, indicating substantial negative potential. When the local electrostatics of the QB site was modulated by a single mutation, L213Asp → Ala, or double mutations, L213Asp-L212Glu → Ala-Ala (AA), the pK of stigmatellin dropped to 7.5 and 7.4, respectively, which corresponds to a >210 mV increase in the electrostatic potential relative to the wild-type RC. This significant pK drop (ΔpK > 3.5) decreased dramatically to (ΔpK > 0.75) in the RC of the compensatory mutant (AA+M44Asn → AA+M44Asp). Our results indicate that the L213Asp is the most important actor in the control of the electrostatic potential in the QB site of the dark-state wild-type RC, in good accordance with conclusions of former studies using theoretical calculations or light-induced charge recombination assay.

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.

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.

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.

M234Glu is a component of the proton sponge in the reaction center from photosynthetic bacteria.

Bacterial reaction centers use light energy to couple the uptake of protons to the successive semi-reduction of two quinones, namely Q(A) and Q(B). These molecules are situated symmetrically in regard to a non-heme iron atom. Four histidines and one glutamic acid, M234Glu, constitute the five ligands of this atom. By flash-induced absorption spectroscopy and delayed fluorescence we have studied in the M234EH and M234EL variants the role played by this acidic residue on the energetic balance between the two quinones as well as in proton uptake. Delayed fluorescence from the P(+)Q(A)(-) state (P is the primary electron donor) and temperature dependence of the rate of P(+)Q(A)(-) charge recombination that are in good agreement show that in the two RC variants, both Q(A)(-) and Q(B)(-) are destabilized by about the same free energy amount: respectively approximately 100 +/- 5 meV and 90 +/- 5 meV for the M234EH and M234EL variants, as compared to the WT. Importantly, in the M234EH and M234EL variants we observe a collapse of the high pH band (present in the wild-type reaction center) of the proton uptake amplitudes associated with formation of Q(A)(-) and Q(B)(-). This band has recently been shown to be a signature of a collective behaviour of an extended, multi-entry, proton uptake network. M234Glu seems to play a central role in the proton sponge-like system formed by the RC protein.

Changes in provider attitudes toward telemedicine.

A longitudinal study was conducted in which the pre- and post-telemedicine encounter attitudes of healthcare providers were compared in order to ascertain whether and how experience with telemedicine changes their attitudes toward telemedicine. Attitudinal changes of providers who had used telemedicine previously were compared to those experiencing telemedicine for the first time. Data were gathered from the providers in two telemedicine programs located in Georgia and Nebraska. Both used real-time videoconferencing and peripheral devices to conduct telemedicine consultations. A total of 87 providers completed questionnaires just prior to and immediately after each telemedicine encounter in their respective programs. The questions focused on the expected impact of telemedicine on their productivity and ability to prescribe treatment. A 3-point scale was used to measure the responses. More than three quarters (79.3%) of the providers did not change their attitudes subsequent to the use of telemedicine. There was no significant difference between first-time users and those who had experience (p = 0.392). The shift in attitude in the minority (n = 18) of providers who did change their minds after the telemedicine encounter was more positive among those who used telemedicine for the first time as compared to those with experience. Contrariwise, those with experience became more negative (p = 0.025). This finding suggests that experience with telemedicine results in more positive attitudes that may not be realized in subsequent interactions with the technology.

Correlating ultrafast function with structure in single crystals of the photosynthetic reaction center.

Femtosecond transient absorbance spectroscopy was applied to the study of primary electron transfer in single reaction center crystals from Rhodobacter sphaeroides. Polarized transient absorption spectra of individual crystals are shown to correlate with polarized ground-state absorption spectra and to track cofactor transition moment directions calculated from the crystallographic structure. Electron transfer from the bacteriochlorophyll dimer to the bacteriopheophytin acceptor was found to be multiphasic in crystals and approximately 2-fold slower than in solution. This work demonstrates the ability to resolve ultrafast photosynthetic function in single crystals and allows ultrafast function to be directly correlated with structure.

Evaluation of the photosynthetic reaction center protein for potential use as a bioelectronic circuit element.

The characterization of a bioelectronic composite prepared by molecular wiring of a bacterial photosynthetic reaction center (RC) to a metal (Au) electrode is described. Two unique attachment sites on the protein surface were studied as sites for electrical connections--a polyhistidine tag introduced by site-directed mutagenesis and a native cysteine amino acid residue. These two attachment sites were evaluated independently and found to serve effectively in coupling the protein to the electrode surface asymmetrically. Cyclic voltammetry (CV) was used to monitor protein integrity and confirm protein chemisorption and orientation to the organofunctionalized gold electrode. Single-protein transport measurements made with conductive atomic force microscopy (C-AFM) were used to study the electrical transport. Current-voltage (I-V) curves obtained by wiring the protein at the polyhistidine tag showed diodelike behavior. The cysteine attachment site does not serve as an efficient means to address the protein electrically. Scanning tunneling spectroscopy (STS) performed on RCs coupled at the donor side under both dark- and white-light-illuminated conditions confirmed the C-AFM studies.

Inactivation of parvovirus B19 in human platelet concentrates by treatment with amotosalen and ultraviolet A illumination.

BACKGROUND: The human erythrovirus B19 (B19) is a small (18- to 26-nm) nonenveloped virus with a single-stranded DNA genome of 5.6 kb. B19 is clinically significant and is also generally resistant to pathogen inactivation methods. Photochemical treatment (PCT) with amotosalen and ultraviolet A (UVA) inactivates viruses, bacteria, and protozoa in platelets (PLTs) and plasma prepared for transfusion. In this study, the capacity of PCT to inactivate B19 in human PLT concentrates was evaluated. STUDY DESIGN AND METHODS: B19 inactivation was measured by a novel enzyme-linked immunosorbent spot (ELISPOT) erythroid progenitor cell infectivity assay and by inhibition of long-range (up to 4.3 kb) polymerase chain reaction (PCR), under conditions where the whole coding region of the viral genome was amplified. B19-infected plasma was used to test whether incubation of amotosalen with virus before PCT enhanced inactivation compared to immediate PCT. RESULTS: Inactivation of up to 5.8 log of B19 as measured by the infectivity assay, or up to 6 logs as measured by PCR inhibition can be achieved under non-limiting conditions. Inactivation efficacy was found to increase with incubation prior to UVA illumination. Without incubation prior to illumination 2.1 +0.4 log was inactivated as determined by infectivity assay. When measured by PCR inhibition, inactivation varied inversely with amplicon size. When primers that spanned the entire coding region of the B19 genome were used, maximum inhibition of PCR amplification was demonstrated. CONCLUSION: Under defined conditions, PCT with amotosalen combined with UVA light can be used to inactivate B19, a clinically significant virus that can be transmitted through blood transfusion, and heretofore has been demonstrated to be refractory to inactivation.

Quantification of viral inactivation by photochemical treatment with amotosalen and UV A light, using a novel polymerase chain reaction inhibition method with preamplification.

BACKGROUND: In evaluating a photochemical treatment process for inactivating parvovirus B19, there lacked simple culture methods to measure infectivity. The recently developed enzyme-linked immunospot (ELISpot) infectivity assay uses late-stage erythropoietic progenitor cells and is labor intensive and time consuming. We evaluated a novel, efficient polymerase chain reaction (PCR) inhibition assay and examined correlations with reductions in infectivity. METHODS: Contaminated plasma was treated with 150 micromol/L amotosalen and 3 J/cm(2) ultraviolet A light and then tested for DNA modification using conventional PCR inhibition and a novel preamplification approach. The novel assay subjected the samples to preamplification cycles using long-template PCR, followed by quantitative PCR (QPCR) inhibition detection. Both approaches were tested for correlations with reductions in viral infectivity by comparing ELISpot assay results of identical samples. RESULTS: The B19 preamplification inhibition assay showed detection ranges of 2-2.5 log and demonstrated quantitative correlation with up to a 5.8-log reduction in viral infectivity in ELISpot results. Conventional PCR detected a >5 log reduction in amplification, correlated with a 4.4-log reduction in viral infectivity. A range of 4-log inhibition of hepatitis B virus DNA amplification was also achieved. CONCLUSIONS: The results demonstrated that a novel preamplification QPCR assay is a useful tool for predicting reductions in infectivity after photochemical treatment. This assay was extended to show utility in circumstances where practical in vitro assays are unavailable for the determination of the efficacy of pathogen inactivation.

Determination of the rate and yield of B-side quinone reduction in Rhodobacter capsulatus reaction centers.

In the native purple bacterial reaction center (RC), light-driven charge separation utilizes only the A-side cofactors, with the symmetry related B-side inactive. The process is initiated by electron transfer from the excited primary donor (P*) to the A-side bacteriopheophytin (P* --> P+ H(A)-) in approximately 3 ps. This is followed by electron transfer to the A-side quinone (P+ H(A)- --> P+ Q(A)-) in approximately 200 ps, with an overall quantum yield of approximately 100%. Using nanosecond flash photolysis and RCs from the Rhodobacter capsulatus F(L181)Y/Y(M208)F/L(M212)H mutant (designated YFH), we have probed the decay pathways of the analogous B-side state P+ H(B)-. The rate of the P+ H(B)- --> ground-state charge-recombination process is found to be (3.0 +/- 0.8 ns)(-1), which is much faster than the analogous (10-20 ns)(-1) rate of P+ H(A)- --> ground state. The rate of P+ H(B)- --> P+ Q(B)- electron transfer is determined to be (3.9 +/- 0.9 ns)(-1), which is about a factor of 20 slower than the analogous A-side process P+ H(A)- --> P+ Q(A)-. The yield of P+ H(B)- --> P+ Q(B)- electron-transfer calculated from these rate constants is 44%. This value, when combined with the known 30% yield of P+ H(B)- from P in YFH RCs, gives an overall yield of 13% for B-side charge separation P* --> P+ H(B)- --> P+ Q(B)- in this mutant. We determine essentially the same value (15%) by comparing the P-bleaching amplitude at approximately 1 ms in YFH and wild-type RCs.