Ronald Pace

A biosensor that uses ion-channel switches

Biosensors are molecular sensors that combine a biological recognition mechanism with a physical transduction technique. They provide a new class of inexpensive, portable instrument that permit sophisticated analytical measurements to be undertaken rapidly at decentralized locations. However, the adoption of biosensors for practical applications other than the measurement of blood glucose is currently limited by the expense, insensitivity and inflexibility of the available transduction methods. Here we describe the development of a biosensing technique in which the conductance of a population of molecular ion channels is switched by the recognition event. The approach mimics biological sensory functions and can be used with most types of receptor, including antibodies and nucleotides. The technique is very flexible and even in its simplest form it is sensitive to picomolar concentrations of proteins. The sensor is essentially an impedance element whose dimensions can readily be reduced to become an integral component of a microelectronic circuit. It may be used in a wide range of applications and in complex media, including blood. These uses might include cell typing, the detection of large proteins, viruses, antibodies, DNA, electrolytes, drugs, pesticides and other low-molecular-weight compounds.

Probing the Mn oxidation states in the OEC. Insights from spectroscopic, computational and kinetic data.

Results from a variety of experimental techniques which have been used to define the oxidation levels of Mn and other components in the S states of the water oxidising complex in Photosystem II are reviewed. A self-consistent interpretation of Mn X-ray absorption near edge spectroscopy, UV-visible and near infrared spectroscopic data suggests that Mn oxidation occurs only on the S0-->S1 transition, and that all four Mn centres have formal oxidation state III thereafter. Ligand oxidation occurs on the transitions to S2 and S3. This is supported by high level quantum chemical calculations and an analysis of the kinetics of substrate water exchange, as recently determined by Wydrzynski et al. (this journal). One type of model for the catalytic site structure and water oxidation mechanism, consistent with these conclusions, is discussed. This model invokes magnetically separate oxo bridged dimers with water oxidation occurring by a concerted 2H+/2e- transfer mechanism, with one H transfer to a bridge oxygen on each dimer.

EPR SATURATION AND TEMPERATURE DEPENDENCE STUDIES ON SIGNALS FROM THE OXYGEN-EVOLVING CENTRE OF PHOTOSYSTEM II

Microwave power saturation studies have been performed over the range 4–20 K on EPR signals photogenerated in PS II particles by low-temperature illumination (180–240 K). In the presence of 3% methanol (+ MeOH), with no g 4.1 signal present, the multiline signal intensity (extrapolated to zero power) shows strict Curie law behaviour over the 4–20 K range. With no MeOH present in the suspension buffer (−MeOH), both the multiline and 4.1 signals show complementary deviations from Curie law behaviour. These are consistent with the signals arising respectively from the ground, S = 1/2, and first excited S = 3/2, states of a total spin = 7/2 multiplet, such as could occur in an MnIV-MnIII antiferromagnetically coupled pair. The deduced height of the 3/2 state above the ½ state is 9.0 K. An inferential estimate, from relaxation data, of this height for the +MeOH case is about 40 K. A broad, featureless component around g = 2 appears to underlie the multiline pattern in the presence of the 4.1 signal, and has a similar temperature behaviour to the latter. A possible exchange coupling model, involving four Mn centres, is presented to accommodate these and other findings on the S2 state signals.

Rationalizing the 1.9 Å Crystal Structure of Photosystem II—A Remarkable Jahn–Teller Balancing Act Induced by a Single Proton Transfer

Green plants and algae oxidize water to molecular oxygen in photosystem II (PS II) within a calcium/tetramanganese site known as the water-oxidizing complex (WOC). Oxygen is generated by the WOC in a four-electron process involving a series of intermediate states (S states, labeled S0...S4) of increasingly higher mean oxidation level. Over the past decade, X-ray crystallographic (XRD) structures of PS II at progressively improved resolution have revealed much detail of the WOC. At present, only PS II from thermophilic cyanobacteria has been crystallized for XRD study and the enzyme is presumed to be in the dark stable S1 state. The first PS II structure (at 3.5 resolution) to resolve side chain positions was presented by Barber and co-workers. Consistent with subsequent studies at higher resolution, it revealed the compact Mn3Ca “cube” structure of the WOC connected more distantly to a single Mn, referred to as the “dangler”. More recent improved structures at 3.0 and 2.9 , substantially clarified the metaland proteinsupplied ligand positions within the WOC, but were still of insufficient resolution to reveal the positions of bridging oxo groups and water molecules (including the substrate water molecules). Finally, Umena et al., using a new crystallization method, produced an atomic resolution structure at 1.9 , the most resolved to date. Despite this remarkable achievement, revealing, for the first time, the positions of bridging O atoms within the Mn4Ca core of the WOC, aspects of the new structure have been met with scepticism. 4] Central concerns over this structure involve 1) the identity and unexpected placement of the O(5) moiety (Figure 1), which appears to be either a weakly bound oxo, hydroxo, or water ligand at distances of 2.4–2.7 from four of the metal atoms in the WOC, and 2) the disparity in some key metal– metal distances when compared with earlier, high-precision extended X-ray absorption fine structure (EXAFS) results and the previous lower-resolution XRD structures (see Table 1). Although the Mn EXAFS data do not unambigu-

Application of computational chemistry to understanding the structure and mechanism of the Mn catalytic site in photosystem II--a review.

Applications of Density Functional Theory (DFT) computational techniques to studies of the molecular structure and mechanism of the oxygen evolving, water oxidising Mn(4)/Ca catalytic site in Photosystem II are reviewed. We summarise results from the earlier studies (pre 2000) but concentrate mainly on those developments which have occurred since publication of several PS II crystal structures of progressively increasing resolution, starting in 2003. The work of all computational groups actively involved in PS II studies is examined, in the light of direct PS II structural information from X-ray diffraction crystallography and EXAFS on the metals in the catalytic site. We further address the consistency of the various computational models with results from a range of spectroscopic studies on the PS II site, in all of those functionally intermediate states (S-states) amenable to study. Experimental data considered include Mn K-edge XANES studies, hyperfine coupling of Mn nuclei and various ligand nuclei (including those from substrate water) seen by several EPR techniques applied to the net spin half intermediates, S(0) and S(2), at low temperatures. Finally we consider proposed catalytic mechanisms for the O-O bond formation step, from two groups, in the light of the available experimental evidence bearing on this process, which we also summarise.

What Are the Oxidation States of Manganese Required To Catalyze Photosynthetic Water Oxidation

Photosynthetic O(2) production from water is catalyzed by a cluster of four manganese ions and a tyrosine residue that comprise the redox-active components of the water-oxidizing complex (WOC) of photosystem II (PSII) in all known oxygenic phototrophs. Knowledge of the oxidation states is indispensable for understanding the fundamental principles of catalysis by PSII and the catalytic mechanism of the WOC. Previous spectroscopic studies and redox titrations predicted the net oxidation state of the S(0) state to be (Mn(III))(3)Mn(IV). We have refined a previously developed photoassembly procedure that directly determines the number of oxidizing equivalents needed to assemble the Mn(4)Ca core of WOC during photoassembly, starting from free Mn(II) and the Mn-depleted apo-WOC complex. This experiment entails counting the number of light flashes required to produce the first O(2) molecules during photoassembly. Unlike spectroscopic methods, this process does not require reference to synthetic model complexes. We find the number of photoassembly intermediates required to reach the lowest oxidation state of the WOC, S(0), to be three, indicating a net oxidation state three equivalents above four Mn(II), formally (Mn(III))(3)Mn(II), whereas the O(2) releasing state, S(4), corresponds formally to (Mn(IV))(3)Mn(III). The results from this study have major implications for proposed mechanisms of photosynthetic water oxidation.

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.

The S-state dependence of Cl− binding to plant Photosystem II

Abstract A combined single-turnover flash and 35 Cl NMR technique has been used to monitor S-state dependence of Cl − binding to PS-II particles derived from mangrove ( Avicennia marina ). No detectable high-affinity binding was found to particles in the S 0 and S 1 states, but binding with an affinity comparable to that which activates O 2 evolution was found in the S 2 and S 3 states.

Structural, Magnetic Coupling and Oxidation State Trends in Models of the CaMn4 Cluster in Photosystem II

Density functional theory calculations are reported on a set of isomeric structures I, II and III that share the structural formula [CaMn4C9H10N2O16]q+.(H2O)3 (q= -1, 0, 1, 2, 3). Species I has a skeletal structure, which has been previously identified as a close match to the ligated CaMn4 cluster in Photosystem II, as characterized in the most recent 3.0 angstroms crystal structure. Structures II and III are rearrangements of I, which largely retain that models bridging ligand framework, but feature metal atom positions broadly consistent with, respectively, the earlier 3.5 and 3.2 angstroms crystal structures for the Photosystem II water-oxidising complex (WOC). Our study explores the influence of the cluster charge state (and hence S state) on several important properties of the model structures; including the relative energies of the three models, their interconversion, trends in the individual Mn oxidation states, preferred hydration sites and favoured modes of magnetic coupling between the manganese atoms. We find that, for several of the explored cluster charge states, modest differences in the bridging-ligand geometry exert a powerful influence over the individual manganese oxidation states, but throughout these states the robustness of the tetrahedron formed by the Ca and three of the Mn atoms remains a significant feature and contrasts with the positional flexibility of the fourth Mn atom. Although structure I is lowest in energy for most S states, the energy differences between structures for a given S state are not large. Overall, structure II provides a better match for the EXAFS derived metal-metal distance parameters for the earlier S states (S0 to S2), but not for S3 in which a significant structural change is observed experimentally. In this S state structure III provides a closer fit. The implications of these results, for the possible action of the WOC, are discussed.

Spectroscopic identification of a dinuclear metal centre in manganese(II)-activated aminopeptidase P from Escherichia coli: implications for human prolidase

Abstract Electron paramagnetic resonance (EPR) spectra and X-ray absorption (EXAFS and XANES) data have been recorded for the manganese enzyme aminopeptidase P (AMPP, PepP protein) from Escherichia coli. The biological function of the protein, a tetramer of 50-kDa subunits, is the hydrolysis of N-terminal Xaa-Pro peptide bonds. Activity assays confirm that the enzyme is activated by treatment with Mn2+. The EPR spectrum of Mn2+–activated AMPP at liquid-He temperature is characteristic of an exchange-coupled dinuclear Mn(II) site, the Mn-Mn separation calculated from the zero-field splitting D of the quintet state being 3.5 (±0.1) Å. In the X-ray absorption spectrum of Mn2+–activated AMPP at the Mn K edge, the near-edge features are consistent with octahedrally coordinated Mn atoms in oxidation state +2. EXAFS data, limited to k≤12 Å–1 by traces of Fe in the protein, are consistent with a single coordination shell occupied predominantly by O donor atoms at an average Mn-ligand distance of 2.15 Å, but the possibility of a mixture of O and N donor atoms is not excluded. The Mn-Mn interaction at 3.5 Å is not detected in the EXAFS, probably due to destructive interference from light outer-shell atoms. The biological function, amino acid sequence and metal-ion dependence of E. coli AMPP are closely related to those of human prolidase, an enzyme that specifically cleaves Xaa-Pro dipeptides. Mutations that lead to human prolidase deficiency and clinical symptoms have been identified. Several known inhibitors of prolidase also inhibit AMPP. When these inhibitors are added to Mn2+–activated AMPP, the EPR spectrum and EXAFS remain unchanged. It can be inferred that the inhibitors either do not bind directly to the Mn centres, or substitute for existing Mn ligands without a significant change in donor atoms or coordination geometry. The conclusions from the spectroscopic measurements on AMPP have been verified by, and complement, a recent crystal structure analysis.