Tom Wydrzynski

On-line mass spectrometry: membrane inlet sampling

Significant insights into plant photosynthesis and respiration have been achieved using membrane inlet mass spectrometry (MIMS) for the analysis of stable isotope distribution of gases. The MIMS approach is based on using a gas permeable membrane to enable the entry of gas molecules into the mass spectrometer source. This is a simple yet durable approach for the analysis of volatile gases, particularly atmospheric gases. The MIMS technique strongly lends itself to the study of reaction flux where isotopic labeling is employed to differentiate two competing processes; i.e., O2 evolution versus O2 uptake reactions from PSII or terminal oxidase/rubisco reactions. Such investigations have been used for in vitro studies of whole leaves and isolated cells. The MIMS approach is also able to follow rates of isotopic exchange, which is useful for obtaining chemical exchange rates. These types of measurements have been employed for oxygen ligand exchange in PSII and to discern reaction rates of the carbonic anhydrase reactions. Recent developments have also engaged MIMS for online isotopic fractionation and for the study of reactions in inorganic systems that are capable of water splitting or H2 generation. The simplicity of the sampling approach coupled to the high sensitivity of modern instrumentation is a reason for the growing applicability of this technique for a range of problems in plant photosynthesis and respiration. This review offers some insights into the sampling approaches and the experiments that have been conducted with MIMS.

Photoproduction of hydrogen peroxide in Photosystem II membrane fragments: A comparison of four signals.

The present study describes the formation of different forms of peroxide in Photosystem II (PS II) by using a chemiluminescence detection technique. Four chemiluminescence signals (A, B, C and D) of the luminolperoxidase (Lu-Per) system, which detects peroxide, are found in illuminated O2-evolving Photosystem II (PS II) membrane fragments isolated from spinach. Signal A (‘free peroxide’) peaking around 0.2–0.3 s after mixing PS II membrane fragments with Lu-Per is eliminated by catalase or removal of oxygen from the suspension and ascribed to O2 interaction with reduced PS II electron acceptors. In contrast, signal B peaking around 1.5 min remains largely unaffected under anaerobic conditions, as well as in the presence of catalase (20 μg/ml). Under flash illumination the extent of this signal exhibits a weak period four oscillation (maximum at third and 7th flash). Its yield increases up to the third flash, but is close to zero in the fourth flash. An analogous behaviour is observed in flashes 5 to 8. Signal B is ascribed to Lu-Per interaction with the water-oxidizing system being in S2 and/or S3-state. Signal C (‘bound peroxide’) detected as free peroxide after acid decomposition of illuminated PS II particles is observed on the 1 st flash and oscillates with period 2 with superposition of period 4. It is evidently related to peroxide either released from S2 or formed at S2 upon acid shock treatment. Signal D (‘slowly released peroxide’) peaking around 2–3 s after mixing is observed in samples after various treatments (LCC-incubation, washing with 1 M NaCl at pH 8 or with 1 M CaCl2, Cl--depletion) that lead to at least partial removal of the extrinsic proteins of 18, 24 and 33 kDa without Mn extraction. The average amplitude of this signal corresponds with a yield of about 0.2 H2O2 molecules per RC and flash. In a flash train, the extent of signal D exhibits an oscillation pattern with a minimum at the 3rd flash. We assume that these treatments increase the release of ‘bound’ peroxide (upon injection into the Lu-Per assay) either formed in the normal oxidative pathway of the water oxidase in the S2 or the S3-state or give rise to peroxide formation due to higher accessibility of the Mn-cluster to water molecules.

Formation and decay of monodehydroascorbate radicals in illuminated thylakoids as determined by EPR spectroscopy

Abstract H 2 O 2 is reduced by an ascorbate-specific peroxidase (APX) in chloroplasts, generating the monodehydroascorbate (MDA) radical as the primary oxidation product. Using EPR spectroscopy we have measured the light-driven formation and decay of this species in thylakoids containing active APX. Illumination caused a rapid exponential rise in the steady-state MDA radical concentration in the absence of added electron acceptors other than O 2 . This increase was sensitive to KCN and catalase and was prevented by anaerobic conditions, demonstrating the requirement for APX activity and endogenously generated H 2 O 2 , i.e., the Mehler reaction. When the illumination was removed, a second, transient increase in the radical signal was observed, indicating that photoreduction of the MDA radical and O 2 were occurring simultaneously in the light. This interpretation is also supported by the sigmoidal behavior of the chlorophyll dependence of MDA radical formation in illuminated thylakoids. Ferredoxin lowered the light-induced, steady-state MDA radical concentration, and is thus implicated as the physiological photoreductant for this Hill acceptor. In the absence of uncoupler, NADP + prevented formation of the MDA radical by lowering the flux to molecular O 2 . However, in the presence of uncoupler (5 mM NH 4 Cl) this constraint was apparently overcome, i.e., net formation of the radical occurred. The EPR method represents a novel approach to investigating the interaction of O 2 and ascorbate metabolism in chloroplasts under a variety of physiologically relevant conditions, to be applied in future studies of plant response to environmental stress.

Increases in peroxide formation by the Photosystem II oxygen evolving reactions upon removal of the extrinsic 16, 22 and 33 kDa proteins are reversed by CaCl2 addition

This communication introduces a new spectrophotometric assay for the detection of peroxide generated by Photosystem II (PS II) under steady state illumination in the presence of an electron acceptor. The assay is based on the formation of an indamine dye in a horseradish peroxidase coupled reaction between 3-(dimethylamino)benzoic acid and 3-methyl-2-benzothiazolinone hydrazone. Using this assay, we found that as the O2 evolution activity of PS II-enriched membrane fragments is decreased by treatments which cause the dissociation of the 33 and/or 23 and 16 kDa extrinsic proteins (i.e., CaCl2-washing, NaCl-washing, lauroylcholine-treatment and ethylene glycol-treatment), light-induced peroxide formation increases. Both the losses of O2 evolution and increases in peroxide formation seen under these conditions are reversed by CaCl2 addition, indicating that the two activities originate from the water-splitting site. However, the increased rates of peroxide formation do not quantitatively match the losses in O2 evolution activity. We suggest that a rapid consumption of the peroxide takes place via a catalase/peroxidase activity at the water-splitting site which competes with both the O2 evolution and peroxide formation reactions. The observed peroxide formation is interpreted as arising from enhanced water accessibility to the catalytic site upon perturbation of the extrinsic proteins which then leads to alternate water oxidation side reactions.

Photo-catalytic oxidation of a di-nuclear manganese centre in an engineered bacterioferritin 'reaction centre'

Photosynthesis involves the conversion of light into chemical energy through a series of electron transfer reactions within membrane-bound pigment/protein complexes. The Photosystem II (PSII) complex in plants, algae and cyanobacteria catalyse the oxidation of water to molecular O2. The complexity of PSII has thus far limited attempts to chemically replicate its function. Here we introduce a reverse engineering approach to build a simple, light-driven photo-catalyst based on the organization and function of the donor side of the PSII reaction centre. We have used bacterioferritin (BFR) (cytochrome b1) from Escherichia coli as the protein scaffold since it has several, inherently useful design features for engineering light-driven electron transport. Among these are: (i.) a di-iron binding site; (ii.) a potentially redox-active tyrosine residue; and (iii.) the ability to dimerise and form an inter-protein heme binding pocket within electron tunnelling distance of the di-iron binding site. Upon replacing the heme with the photoactive zinc-chlorin e6 (ZnCe6) molecule and the di-iron binding site with two manganese ions, we show that the two Mn ions bind as a weakly coupled di-nuclear Mn2II,II centre, and that ZnCe6 binds in stoichiometric amounts of 1:2 with respect to the dimeric form of BFR. Upon illumination the bound ZnCe6 initiates electron transfer, followed by oxidation of the di-nuclear Mn centre possibly via one of the inherent tyrosine residues in the vicinity of the Mn cluster. The light dependent loss of the MnII EPR signals and the formation of low field parallel mode Mn EPR signals are attributed to the formation of MnIII species. The formation of the MnIII is concomitant with consumption of oxygen. Our model is the first artificial reaction centre developed for the photo-catalytic oxidation of a di-metal site within a protein matrix which potentially mimics water oxidation centre (WOC) photo-assembly.

Creating Functional Artificial Proteins

Much is now known about how protein folding occurs, through the sequence analysis of proteins of known folding geometry and the sequence/structural analysis of proteins and their mutants. This has allowed not only the modification of natural proteins but also the construction of de novo polypeptides with predictable folding patterns. Structure/function analysis of natural proteins is used to construct derived versions that retain a degree of biological activity. The constructed versions made of either natural or artificial sequences contain critical residues for activity such as receptor binding. In some cases, the functionality is introduced by incorporating binding sites for other elements, such as organic cofactors or transition metals, into the protein scaffold. While these modified proteins can mimic the function of natural proteins, they can also be constructed to have novel activities. Recently engineered photoactive proteins are good examples of such systems in which a light-induced electron transfer can be established in normally light-insensitive proteins. The present review covers some aspects of protein design that have been used to investigate protein receptor binding, cofactor binding and biological electron transfer.

A Fourier transform infrared spectroscopic study of the effects of hydrogen peroxide and high light on protein conformations of Photosystem II

Degradation of the reaction center-binding protein D1 of Photosystem II (PS II) during photoinhibition is dependent on the action of active oxygen species and/or D1-specific proteases. Protein conformational changes may be involved in the process of D1 degradation. In the present study, we determined the effect of H2O2 on spinach PS II-enriched membranes and core complexes with respect to electron transport, Mn content and protein secondary structural changes as measured by Fourier transform infrared (FTIR) spectroscopy. H2O2 is effective in removing catalytic Mn in PS II, especially in PS II core complexes depleted of OEC18 and OEC24, impairing the donor-side. By quantitative analysis of the amide I band (1600 – 1700 cm-1) with both aqueous and dehydrated PS II samples, we found that no significant secondary structural changes are associated with H2O2 treatment in the dark, even though there is some cleavage of the D1 protein by H2O2 treatment as determined by Western analysis with specific antibodies. In contrast, a large decrease in the α-helices in the PS II core occurs, with or without H2O2 treatment, after 20 min strong illumination and there is more extensive degradation of the D1 protein. Our results suggest that high light enhances the cleavage of the D1 protein which is reflected in the large protein secondary structural changes in PS II detected by FTIR measurements.

Amino acid deletions in the cytosolic domains of the chlorophyll a-binding protein CP47 slow Q(A)- oxidation and/or prevent the assembly of photosystem II.

The Photosystem II (PSII) core antenna chlorophyll a-binding protein, CP47, contains six membrane-spanning α-helices separated by five hydrophilic loops: A–E. To identify important hydrophilic cytosolic regions, oligonucleotide-directed mutagenesis was employed to introduce short segment deletions into loops B and D, and the C-terminal domain. Four strains carrying deletions of between three and five residues were created in loop B. Two strains, with deletions adjacent to helices II and III, did not assemble PSII; however, the mutants Δ(F123–D125) and Δ(R127–S131) remained photoautotrophic with near wild-type levels of assembled reaction centers. In contrast, all deletions introduced into loop D, connecting helices IV and V, failed to assemble significant levels of PSII and were obligate photoheterotrophic mutants. However, deletions in the C-terminal domain did not prevent the assembly of PSII reaction centers although the mutant Δ(S471–T473), with a deletion adjacent to helix VI, exhibited retarded QA− oxidation kinetics and the PSII-specific herbicide, atrazine, bound less tightly in the Δ(S471–T473) and Δ(F475–D477) strains. Deletions in the C-terminal domain also created mutants with large protein aggregates that were recognized by an antibody raised against the PSII reaction center D1 protein. Low-temperature fluorescence emission spectra of photoautotrophic strains carrying deletions in either the C-terminal domain or loop B did not provide evidence for impaired energy transfer from the phycobilisomes to the PSII reaction center. The data therefore suggest an important structural role for loop D in the assembly of PSII and a potential interaction between the C-terminal domain of CP47 and the PSII reaction center that, when perturbed, results in photoinduced protein aggregates involving the D1 protein.

Substrate Water 18 O Exchange Kinetics in the S 2 State of Photosystem II

The oxidation of water into molecular oxygen during photosynthesis is catalyzed by a subdomain of photosystem II (PSII) termed the water oxidizing complex (WOC). Contained within the WOC is a redox-active Mn/tyrosine motif which forms, in part, the catalytic site. Fundamental to the water oxidation mechanism is the periodicity of four in the release of O2 upon flash illumination. This unique behavior can readily be explained by a phenomenological model in which the WOC cycles through five intermediate states called the S-states (or Sn where n=0-4). Upon attaining the metastable S4 state, O2 is released, the S0 state is regenerated, and the S-state cycle begins again [1].

Chapter 16:Synthetic Photo-catalytic Proteins – a Model of Photosystem II

A primary goal in solar fuels research is the development of an efficient photo-catalyst that splits water into molecular O2 and H2 using solar energy. Uniquely, Nature almost achieved this goal some 2.5 billion years ago by separating the two half-reactions for O2 and H2 production from water into different protein complexes: Photosystem II (PSII) in higher plants and a H+-reducing enzyme (HRE) found in various micro-organisms. In this chapter we briefly summarize some of the bioengineering principles for developing a photo-catalytic protein and describe our first efforts to mimic PSII.