Near-IR irradiation of the S2 state of the water oxidizing complex of photosystem II at liquid helium temperatures produces the metalloradical intermediate attributed to S1Y(Z*)

Near-IR (NIR) excitation at liquid He temperatures of photosystem II (PSII) membranes from the cyanobacterium Synechococcus vulcanus or from spinach poised in the S2 state results in the production of a g = 2.035 EPR resonance, reminiscent of metalloradical signals. The signal is smaller in the spinach preparations, but it is significantly enhanced by the addition of exogenous quinones. Ethanol (2-3%, v/v) eliminates the ability to trap the signal. The g = 2.035 signal is identical to the one recently obtained by Nugent et al. by visible-light illumination of the S1 state, and preferably assigned to S1Y(Z*) [Nugent, J. H. A., Muhiuddin, I. P., and Evans, M. C. W. (2002) Biochemistry 41, 4117-4126]. The production of the g = 2.035 signal by liquid He temperature NIR excitation of the S2 state is paralleled by a significant reduction (typically 40-45% in S. vulcanus) of the S2 state multiline signal. This is in part due to the conversion of the Mn cluster to higher spin states, an effect documented by Boussac et al. [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007], and in part due to the conversion to the g = 2.035 configuration. Following the decay of the g = 2.035 signal at liquid helium temperatures (decay halftimes in the time range of a few to tens of minutes depending on the preparation), annealing at elevated temperatures (-80 degrees C) results in only partial restoration of the S2 state multiline signal. The full size of the signal can be restored by visible-light illumination at -80 degrees C, implying that during the near-IR excitation and subsequent storage at liquid helium temperatures recombination with Q(A-) (and therefore decay of the S2 state to the S1 state) occurred in a fraction of centers. In support of this conclusion, the g = 2.035 signal remains stable for several hours (at 11 K) in centers poised in the S2...Q(A) configuration before the NIR excitation. The extended stability of the signal under these conditions has allowed the measurement of the microwave power saturation and the temperature dependence in the temperature range of 3.8-11 K. The signal intensity follows Curie law temperature dependence, which suggests that it arises from a ground spin state, or a very low-lying excited spin state. The P1/2 (microwave power at half-saturation) value is 1.7 mW at 3.8 K and increases to 96 mW at 11 K. The large width of the g = 2.035 signal and its relatively fast relaxation support the assignment to a radical species in the proximity of the Mn cluster. The whole phenomenology of the g = 2.035 signal production is analogous to the effects of NIR excitation on the S3 state [Ioannidis, N., Nugent, J. H. A., and Petrouleas, V. (2002) Biochemistry 41, 9589-9600] producing an S2'Y(Z*) intermediate. In the present case, the intermediate is assigned to S1Y(Z*). The NIR-induced increase in the oxidative capability of the Mn cluster is discussed in relation to the photochemical properties of a Mn(III) ion that exists in both S2 and S3 states. The EPR properties of the S1Y(Z*) intermediate cannot be reconciled easily with our current understanding of the magnetic properties of the S1 state. It is suggested that oxidation of tyr Z alters the magnetic properties of the Mn cluster via exchange of a proton.     

Probing tyrosine Z oxidation in photosystem II core complex isolated from spinach by EPR at liquid helium temperatures

Tyrosine Z (Tyr_Z) oxidation observed at liquid helium temperatures provides new insights into the structure and function of Tyr_Z in active Photosystem II (PSII). However, it has not been reported in PSII core complex from higher plants. Here, we report Tyr_Z oxidation in the S₁ and S₂ states in PSII core complex from spinach for the first time. Moreover, we identified a 500 G-wide symmetric EPR signal (peak position g = 2.18, trough position g = 1.85) together with the g = 2.03 signal induced by visible light at 10 K in the S₁ state in the PSII core complex. These two signals decay with a similar rate in the dark and both disappear in the presence of 6% methanol. We tentatively assign this new feature to the hyperfine structure of the S₁Tyr_Z ^• EPR signal. Furthermore, EPR signals of the S₂ state of the Mn-cluster, the oxidation of the non-heme iron, and the S₁Tyr_Z ^• in PSII core complexes and PSII-enriched membranes from spinach are compared, which clearly indicate that both the donor and acceptor sides of the reaction center are undisturbed after the removal of LHCII. These results suggest that the new spinach PSII core complex is suitable for the electron transfer study of PSII at cryogenic temperatures.     

UDPglucose dehydrogenase. Kinetics and their mechanistic implications.

Initial velocity and product inhibition studies were carried out on UDP-glucose dehydrogenase (UDPglucose: NAD+ 6-oxidoreductase, EC 1.1.1.22) from beef liver to determine if the kinetics of the reaction are compatible with the established mechanism. An intersecting initial velocity pattern was observed with NAD+ as the variable substrate and UDPG as the changing fixed substrate. UDPglucuronic acid gave competitive inhibition of UDPG and non-competitive inhibition of NAD+. Inhibition by NADH gave complex patterns.Lineweaver-Burk plots of 1/upsilon versus 1/NAD+ at varied levels of NADH gave highly non-linear curves. At levels of NAD+ below 0.05 mM, non-competitive inhibition patterns were observed giving parabolic curves. Extrapolation to saturation with NAD+ showed NADH gave linear uncompetitive inhibition of UDPG if NAD+ was saturating. However, at levels of NAD+ above 0.10 mM, NADH became a competitive inhibitor of NAD+ (parabolic curves) and when NAD+ was saturating NADH gave no inhibition of UDPG. NADH was non-competitive versus UDPG when NAD+ was not saturating. These results are compatible with a mechanism in which UDPG binds first, followed by NAD+, which is reduced and released. A second mol of NAD+ is then bound, reduced, and released. The irreversible step in the reaction must occur after the release of the second mol of NADH but before the release of UDPglucuronic acid. This is apparently caused by the hydrolysis of a thiol ester between UDPglucoronic acid and the essential thiol group of the enzyme. Examination of rate equations indicated that this hydrolysis is the rate-limiting step in the overall reaction. The discontinuity in the velocities observed at high NAD+ concentrations is apparently caused by the binding of NAD+ in the active site after the release of the second mol of NADH, eliminating the NADH inhibition when NAD+ becomes saturating.     

Dose tolerance at helium and nitrogen temperatures for whole cell electron tomography

Electron tomography is currently the only method that allows the direct three-dimensional visualization of macromolecules in an unperturbed cellular context. In principle, tomography should enable the identification and localization of the major macromolecular complexes within intact bacteria, embedded in amorphous ice. In an effort to optimize conditions for recording data that would bring us close to the theoretical limits, we present here a comparison of the dose tolerance of Caulobacter crescentus cells embedded in amorphous ice at liquid helium versus liquid nitrogen temperature. The inner and outer cell membranes, and the periodic structure of the S-layer of this Gram-negative bacterium provide ideal features to monitor changes in contrast and order as a function of dose. The loss of order in the S-layer occurs at comparable doses at helium and nitrogen temperatures. Macroscopic bubbling within the cell and the plastic support develops at both temperatures, but more slowly at helium temperature. The texture of the bubbles is finer in initial stages at helium temperature, giving an impression of contrast reversal in some parts of the specimen. Bubbles evolve differently in different organelles, presumably a consequence of their different chemical composition and mechanical properties. Finally, the amorphous ice "flows" at helium temperature, causing changes in the relative positions of markers within the specimen and distorting the cells. We conclude that for cryo-electron tomography of whole cells liquid nitrogen temperature provides better overall data quality.     

Chytrids cannot survive at high temperatures in liquid growth media: implications for soil ecosystems

Sixteen isolates in the orders Blastocladiales, Chytridiales, Rhizophydiales and Spizellomycetales were incubated for 2 d in liquid PYG growth medium at 33, 37, 40, 45 and 50 °C. These fungi could not resume growth when returned to 20 °C if the temperature of incubation was more than a few degrees above the maximum temperature for growth on solid PYG growth medium. The maximum temperatures for survival of some chytrids are probably close to the temperatures reached periodically on the surface of moist soils in warm climates. Desiccated thalli of chytrids in the Blastocladiales and Spizellomycetales can survive much higher temperatures than chytrids in the Chytridiales and Rhizophydiales. Therefore, the maximum temperature and moisture content of the soil could significantly affect the diversity of chytrids in the ecosystem.     

Trapping and characterization of covalent intermediates of mutant retaining glycosyltransferases

The enzymatic mechanism by which retaining glycosyltransferases (GTs) transfer monosaccharides with net retention of the anomeric configuration has, so far, resisted elucidation. Here, direct detection of covalent glycosyl-enzyme intermediates for mutants of two model retaining GTs, the human blood group synthesizing α-(1 → 3)-N-acetylgalactosaminyltransferase (GTA) and α-(1 → 3)-galactosyltransferase (GTB) mutants, by mass spectrometry (MS) is reported. Incubation of mutants of GTA or GTB, in which the putative catalytic nucleophile Glu(303) was replaced with Cys (i.e. GTA(E303C) and GTB(E303C)), with their respective donor substrate results in a covalent intermediate. Tandem MS analysis using collision-induced dissociation confirmed Cys(303) as the site of glycosylation. Exposure of the glycosyl-enzyme intermediates to a disaccharide acceptor results in the formation of the corresponding enzymatic trisaccharide products. These findings suggest that the GTA(E303C) and GTB(E303C) mutants may operate by a double-displacement mechanism.     

Protocatechuate 3,4-dioxygenase. Inhibitor studies and mechanistic implications.

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) from Pseudomonas aeruginosa catalyzes the cleavage of 3,4-dihydroxybenzoate (protocatechuate) into beta-carboxy-cis,cis-muconate. The inhibition constants, Ki, of a series of substrate analogues were measured in order to assess the relative importance of the various functional groups on the substrate. Though important for binding, the carboxylate group is not essential for activity. Compounds with para hydroxy groups are better inhibitors than their meta isomers. Our studies of the enzyme-inhibitor complexes indicate that the 4-OH group of the substrate binds to the active-site iron. Taken together, M?ssbauer, EPR, and kinetic data suggest a mechanism where substrate reaction with oxygen is preceded by metal activation of substrate.     

Spectroscopic characterization of true enzyme-substrate intermediates of aspartate aminotransferase trapped at subzero temperatures

Absorption and circular dichroism spectra of stable enzyme-substrate intermediates of aspartate aminotransferase were recorded at subzero temperatures (down to -65 degrees C) in the cryosolvent water/methanol. The intermediates were formed either between the pyridoxal form of the enzyme and its amino acid substrates, or between the pyridoxamine form and its oxo acid substrates. Kd values determined by spectroscopic titration were very close to the Km values reported for the different substrates. The adsorption complex of the pyridoxal form was probably obtained on addition of cysteine sulfinate. This complex is characterized by an increased absorption at 430 nm together with a positive Cotton effect, as also observed in the case of the complex with the competitive inhibitor maleate indicating protonation of the internal aldimine. Addition of the substrates aspartate or glutamate to the pyridoxal form seemed to result in the direct accumulation of the external aldimine which showed a slight decrease in both the absorbance and the Cotton effect at 360 nm. Additionally, a bathochromic shift of 5 nm was observed in the case of glutamate. At 430 nm, only a minor increase in absorbance, but not in circular dichroism, was observed with aspartate, and no changes were found with glutamate and the substrate analog 2-methylaspartate, indicating a deprotonated external aldimine. Presumably, the ketimine intermediate was obtained on addition of the oxo acids 2-oxoglutarate or oxalacetate to the pyridoxamine form. The intermediate showed a slight bathochromic shift (2 nm) of the absorption band and decreased circular dichroism. On formation of the ketimine, a tyrosine residue, probably active-site Tyr225, becomes partly ionized. The finding that the external aldimine can probably be accumulated in the conversion of the pyridoxal to the pyridoxamine form with the natural substrates would confirm the proton abstraction at C alpha to be the rate-limiting step in the tautomerization, although with cysteine sulfinate, the formation of the external aldimine might contribute to the rate limitation. Accumulation of the ketimine in the reverse direction would indicate that the proton abstraction at C4' is rate-limiting in this half-reaction. The results demonstrate the feasibility of further structural investigations of true enzyme-substrate intermediates.     

Absorption spectra of intermediates of bacteriorhodopsin measured by laser photolysis at room temperatures

Picosecond laser spectroscopic analysis was applied to determine how many intermediates existed in the primary photochemical process of trans-bacteriorhodopsin (light-adapted bacteriorhodopsin) at room temperature (18°C) and to calculate their absorption spectra. Irradiation of bacteriorhodopsin with a laser pulse (wavelength, 532 nm; pulse width, 25 ps) yielded the K intermediate (K) which was produced through a precursor, having an absorption maximum (λ_max) longer than that of K. K was stable during a picosecond time range (50–900 ps). The λ_max was located at 610 nm and the extinction coefficient (?_max) was 0.92-times that of bacteriorhodopsin. The same K intermediate was produced from bacteriorhodopsin even when it was excited with a high-energy pulse by which a saturation effect was induced. A transient difference spectrum measured at 150 ns after the excitation of bacteriorhodopsin was different in shape from that of the K intermediate, suggesting that an intermediate was formed by thermal decay of K. This intermediate, tentatively called the KL intermediate (KL), had a λ_max at 596 nm and an ?_max 0.80-times that of bacteriorhodopsin. KL decayed to the L intermediate (L) with a time constant of 2.2 μs. L has a λ_max at 543 nm and an ?_max 0.66-times that of bacteriorhodopsin.     

Crystal structure of rabbit cytosolic serine hydroxymethyltransferase at 2.8 A resolution: mechanistic implications.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to form glycine and single carbon groups that are essential for many biosynthetic pathways. SHMT requires both pyridoxal phosphate (PLP) and tetrahydropteroylpolyglutamate (H4PteGlun) as cofactors, the latter as a carrier of the single carbon group. We describe here the crystal structure at 2.8 A resolution of rabbit cytosolic SHMT (rcSHMT) in two forms: one with the PLP covalently bound as an aldimine to the Nepsilon-amino group of the active site lysine and the other with the aldimine reduced to a secondary amine. The rcSHMT structure closely resembles the structure of human SHMT, confirming its similarity to the alpha-class of PLP enzymes. The structures reported here further permit identification of changes in the PLP group that accompany formation of the geminal diamine complex, the first intermediate in the reaction pathway. On the basis of the current mechanism derived from solution studies and the properties of site mutants, we are able to model the binding of both the serine substrate and the H4PteGlun cofactor. This model explains the properties of several site mutants of SHMT and offers testable hypotheses for a more detailed mechanism of this enzyme.     

In vitro toxicology of fumonisins and the mechanistic implications

The effects of fumonisins B₁FB₁, B₂(FB{2}), and the backbone of fumonisin B₁ remaining after hydrolysis of the tricarballylic groups with base (HFB₁) on sphingolipid biosynthesis were studied in both primary rat hepatocytes and pig kidney epithelial cells (LLC-PK₁). Fumonisins were potent inhibitors of sphingolipid biosynthesis in hepatocytes (IC₅₀ of FB₁=0.1 M), but overt toxicity was not observed. In renal cells, fumonisins also inhibited sphingosine biosynthesis (IC₅₀ for FB₁=35 M), and caused decreased cell proliferation as well. Higher doses (70 M) killed renal cells after exposure for 3 days. The inhibition of de novo sphingolipid biosynthesis was specific, and appeared to be at the site of ceramide synthase, which catalyzes the formation of dihydroceramide or ceramide by the addition of the amide-linked fatty acid to sphinganine or sphingosine. These results may account for the ability of fumonisins to cause equine leucoencephalomalacia and to promote tumor formation.     

Circular dichroism of cattle rhodopsin and bathorhodopsin at liquid nitrogen temperatures

The photoevent in vision has been considered to be the conversion of rhodopsin to bathorhodopsin, which is caused by photoisomerization of the chromophoric retinal. Recently some objections were raised to this hypothesis. The reliability of the hypothesis was verified by measurement of circular dichroism of bathorhodopsin.The measurement of circular dichroism of rhodopsin extract (containing 66% or 75% of glycerol) at liquid nitrogen temperatures (?195°C) by a conventional spectropolarimeter induced an extraordinary large signal, owing to linear dichroism originated from conversion of rhodopsin to bathorhodopsin by the measuring light. The similar linear dichroism can be induced by irradiation of rhodopsin extract at ?195°C with polarized light or natural light. At photosteady state the linear dichroism disappeared.Circular dichroism spectrum of cattle rhodopsin displayed two positive peaks ([θ]_max = 80 800 degrees at 335 nm, and [θ]_max = 42 600 degrees at 500 nm) at ?195°C, whereas, bathorhodopsin displayed a positive peak ([θ]_max = 43 100 degrees at 334 nm) and a negative peak ([θ]_max = 163 000 degrees at 540 nm).The change of the positive sign to negative one at α-band of circular dichroism spectrum supports the hypothesis that the conversion of rhodopsin is due to rotation of the chromophoric retinal about C-11—12 double bond (‘photoisomerization model’).     

EPR studies on the photoinduced intermediates of NO complexes in recombinant ferric-Mb trapped at low temperatures

The nitrosyl complex of ferric myoglobin is EPR-silent. Upon photolysis at low temperatures, the photoinduced intermediates trapped in the distal heme cavity exhibit new EPR spectra due to the interaction between the photodissociated NO (S=1/2) and the ferric high spin heme (S=5/2). In order to elucidate the effect of distal E7 (His64) and E11 (Val68) mutations upon the electronic structure of the metal center, its immediate environment, and its interaction with the photodissociated NO, EPR spectra of the photoproducts of the NO complexes of recombinant ferric Mb mutants were measured at 5 K. EPR spectra of the photoproducts were closely related to the size and/or the polarity of the distal pocket residues. The distal pocket of the E7 mutants seemed to be sterically crowded, even decreasing the side chain volume or changing its hydrophobicity by replacing amino acid at position 64. We have found that the mobility of the photodissociated NO molecule in the distal heme pocket was strongly governed by the nature of the amino acid residue at E11 position.     

Molecular structures of human argininosuccinate synthetase pseudogenes. Evolutionary and mechanistic implications

In the human genome there is one expressed gene for argininosuccinate synthetase and 14 pseudogenes. A cDNA coding for human argininosuccinate synthetase was used to screen a human genomic library. Twenty-five unique genomic clones were isolated and extensively characterized. At least seven clones represented processed argininosuccinate synthetase pseudogenes that lost the introns in the expressed gene. Restriction mapping demonstrated that these processed pseudogenes were located in distinct regions of the human genome. Complete nucleotide sequences of two processed pseudogenes, psi AS-1 and psi AS-3, and a partial sequence of psi AS-7 were determined. Both psi AS-1 and psi AS-3 had an adenine-rich region at their 3' end and were flanked by distinct imperfect direct repeats. A comparison of these pseudogene sequences to that of the cDNA demonstrated that psi AS-1 and psi AS-3 were 93% homologous to the cDNA, whereas psi AS-7 was 89% homologous to the cDNA. Therefore, it is estimated that psi AS-1 and psi AS-3 were created 10-11 million years ago, whereas psi AS-7 arose approximately 21 million years ago. We have estimated the evolutionary rate for the expressed argininosuccinate synthetase gene based on the sequences of psi AS-1 and psi AS-3. These data indicate that the expressed argininosuccinate synthetase gene is evolving at a rate similar to that of the beta-globin gene and much faster than the alpha-tubulin gene. Furthermore, a comparison of the sequences of psi AS-1 and psi AS-3 suggests the possibility that these pseudogenes arose from a common intermediate.