Chemical and structural disorder in eumelanins: A possible explanation for broadband absorbance

We report the results of an experimental and theoretical study of the electronic and structural properties of a key eumelanin precursor-5,6,-dihydroxyindole-2-carboxylic acid ( DHICA) - and its dimeric forms. We have used optical spectroscopy to follow the oxidative polymerization of DHICA to eumelanin and observe red shifting and broadening of the absorption spectrum as the reaction proceeds. First principles density functional theory calculations indicate that DHICA oligomers ( possible reaction products of oxidative polymerization) have the gap between the highest occupied molecular orbital and the lowest unoccupied molecular orbital red-shifted gaps with respect to the monomer. Furthermore, different bonding configurations ( leading to oligomers with different structures) produce a range of gaps. These experimental and theoretical results lend support to the chemical disorder model where the broadband monotonic absorption characteristic of all melanins is a consequence of the superposition of a large number of nonhomogeneously broadened Gaussian transitions associated with each of the components of a melanin ensemble. These results suggest that the traditional model of eumelanin as an amorphous organic semiconductor is not required to explain its optical properties and should be thoroughly reexamined. These results have significant implications for our understanding of the physics, chemistry, and biological function of these important biological macromolecules. Indeed, one may speculate that the robust functionality of melanins in vitro is a direct consequence of its heterogeneity, i.e., chemical disorder is a "low cost" natural resource in these systems

Ultraviolet absorbance of the mucus of a tropical damselfish: Effects of ontogeny, captivity and disease

The ultraviolet (UV) absorbance of the mucus of a Great Barrier Reef damselfish Pomacentrus amboinensis was investigated with regard to ontogeny and time spent in captivity. The UV absorbance of P. amboinensis mucus increased with fish size and decreased with time spent in captivity. The wavelength of maximum absorbance of the mucus did not change with fish size, but shifted towards shorter wavelengths with increasing time spent in captivity. The UV absorbance of the mucus of fish with 'fin rot' was compared to that of similar healthy individuals, and a significant decrease in UV absorbance of unhealthy fish mucus was detected; no wavelength shifting occurred. Pomacentrus amboinensis appears to sequester mycosporine-like amino acids from the diet in order to protect epithelial tissues from UV damage, and decreases in UV absorbance in captive fish were probably due to insufficient dietary availability.

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

The albA gene from Klebsiella oxytoca encodes a protein that binds albicidin phytotoxins and antibiotics with high affinity. Previously, it has been shown that shifting pH from 6 to 4 reduces binding activity of AlbA by about 30%, indicating that histidine residues might be involved in substrate binding. In this study, molecular analysis of the albA coding region revealed sequence discrepancies with the albA sequence reported previously, which were probably due to sequencing errors. The albA gene was subsequently cloned from K oxytoca ATCC 13182(T) to establish the revised sequence. Biochemical and molecular approaches were used to determine the functional role of four histidine residues (His(78), HiS(125), HiS(141) and His(189)) in the corrected sequence for AlbA. Treatment of AlbA with diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, reduced binding activity by about 95%. DEPC treatment increased absorbance at 240-244 nm by an amount indicating conversion to N-carbethoxyhistidine of a single histidine residue per AlbA molecule. Pretreatment with albicidin protected AlbA against modification by DEPC, with a 1 : 1 molar ratio of albicidin to the protected histidine residues. Based on protein secondary structure and amino acid surface probability indices, it is predicted that HiS125 might be the residue required for albicidin binding. Mutation of HiS125 to either alanine or leucine resulted in about 32% loss of binding activity, and deletion of HiS125 totally abolished binding activity. Mutation of HiS125 to arginine and tyrosine had no effect. These results indicate that HiS125 plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.

Endurance exercise, plasma oxidation and cardiovascular risk

Objective-Although physical activity is beneficial to health, people who exercise at high intensities throughout their lifetime may have increased cardiovascular risk. Aerobic exercise increases oxidative stress and may contribute to atherogenesis by augmented oxidation of plasma lipoproteins. The aim of this study was to examine the relationship between aerobic power and markers of oxidative stress, including the susceptibility of plasma to oxidation. Methods and results-Aerobic power was measured in 24 healthy men aged 29 9 years (mean +/- SD). Plasma was analysed from subjects of high aerobic power (HAP; VO(2)max, 64.6 +/- 6.1 ml/kg/min) and lower aerobic power (LAP;VO(2)max, 45.1 +/- 6.3 ml/kg/min) for total antioxidant capacity (TAC), malondialdehyde (MDA) and susceptibility to oxidation. Three measures were used to quantify plasma oxidizability: (1) lag time to conjugated diene formation (lag time); (2) change in absorbance at 234 nm and; (3) slope of the oxidation curve during propagation (slope). The HAP subjects had significantly lowerTAC (1.38 +/- 0.04 versus 1.42 +/- 0.06 TEAC units; P < 0.05), significantly higher change in absorbance (1.55 +/- 0.21 versus 1.36 +/- 0.17 arbitrary units; P < 0.05), but no difference in MDA (P = 0.6), compared to LAP subjects. There was a significant inverse association between TAC and slope (r = -0.49; P < 0.05). Lipoprotein profiles and daily intake of nutrients did not differ between the groups. Conclusions-These findings suggest that people with high aerobic power, due to extreme endurance exercise, have plasma with decreased antioxidant capacity and higher susceptibility to oxidation, which may increase their cardiovascular risk.

Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.

Microspectrophotometry of visual pigments and oil droplets in a marine bird, the wedge-tailed shearwater Puffinus pacificus: Topographic variations in photoreceptor spectral characteristics

Microspectrophotometric examination of the retina of a procellariiform marine bird, the wedge-tailed shearwater Puffinus pacificus, revealed the presence of five different types of vitamin A(1)-based visual pigment in seven different types of photoreceptor. A single class of rod contained a medium-wavelength sensitive visual pigment with a wavelength of maximum absorbance (lambda(max)) at 502 nm. Four different types of single cone contained visual pigments maximally sensitive in either the violet (VS, lambda(max) 406 nm), short (SWS, lambda(max) 450 nm), medium (MWS, lambda(max) 503 nm) or long (LWS, lambda(max) 566 nm) spectral ranges. In the peripheral retina, the SWS, MWS and LWS single cones contained pigmented oil droplets in their inner segments with cut-off wavelengths (lambda(cut)) at 445 (C-type), 506 (Y-type) and 562 nm (R-type), respectively. The VS visual pigment was paired with a transparent (T-type) oil droplet that displayed no significant absorption above at least 370 run. Both the principal and accessory members of the double cone pair contained the same 566 nm lambda(max) visual pigment as the LWS single cones but only the principal member contained an oil droplet, which had a lambda(cut) at 413 nm. The retina had a horizontal band or 'visual streak' of increased photoreceptor density running across the retina approximately 1.5 mm dorsal to the top of the pecten. Cones in the centre of the horizontal streak were smaller and had oil droplets that were either transparent/colourless or much less pigmented than at the periphery. It is proposed that the reduction in cone oil droplet pigmentation in retinal areas associated with high visual acuity is an adaptation to compensate for the reduced photon capture ability of the narrower photoreceptors found there. Measurements of the spectral transmittance of the ocular media reveal that wavelengths down to at least 300 nm would be transmitted to the retina.

Time-resolved spectroscopy of the metal-to-metal charge transfer excited state in dinuclear cyano-bridged mixed-valence complexes

Visible pump-probe spectroscopy has been used to identify and characterize short-lived metal-to-metal charge transfer (MMCT) excited states in a group of cyano-bridged mixed-valence complexes of the formula [(LCoNCMII)-N-III(CN)(5)](-), where L is a pentadentate macrocyclic pentaamine (L-14) or triamine-dithiaether (L-14S) and M is Fe or Ru. Nanosecond pump-probe spectroscopy on frozen solutions of [(LCoNCFeII)-Co-14-N-III(CN)(5)](-) and [(LCoNCFeII)-Co-14S-N-III(CN)(5)](-) at 11 K enabled the construction of difference transient absorption spectra that featured a rise in absorbance in the region of 350-400 nm consistent with the generation of the ferricyanide chromophore of the photoexcited complex. The MMCT excited state of the Ru analogue [(LCoNCRuII)-Co-14-N-III(CN)(5)](-) was too short-lived to allow its detection. Femtosecond pump-probe spectroscopy on aqueous solutions of [(LCoNCFeII)-Co-14-N-III(CN)(5)](-) and [(LCoNCFeII)-Co-14S-N-III(CN)(5)](-) at room temperature enabled the lifetimes of their Co-II-Fe-III MMCT excited states to be determined as 0.8 and 1.3 ps, respectively.

Cone topography and spectral sensitivity in two potentially trichromatic marsupials, the quokka (Setonix brachyurus) and quenda (Isoodon obesulus)

The potential for trichromacy in mammals, thought to be unique to primates, was recently discovered in two Australian marsupials. Whether the presence of three cone types, sensitive to short- (SWS), medium-(MWS) and long-(LWS) wavelengths, occurs across all marsupials remains unknown. Here, we have investigated the presence, distribution and spectral sensitivity of cone types in two further species, the quokka (Setonix brachyurus) and quenda (Isoodon obesulus). Immunohistochemistry revealed that SWS cones in the quokka are concentrated in dorso-temporal retina, while in the quenda, two peaks were identified in naso-ventral and dorso-temporal retina. In both species, MWS/LWS cone spatial distributions matched those of retinal ganglion cells. Microspectrophotometry (MSP) confirmed that MWS and LWS cones are spectrally distinct, with mean wavelengths of maximum absorbance at 502 and 538 nm in the quokka, and at 509 and 551 nm, in the quenda. Although small SWS cone outer segments precluded MSP measurements, molecular analysis identified substitutions at key sites, accounting for a spectral shift from ultraviolet in the quenda to violet in the quokka. The presence of three cone types, along with previous findings in the fat-tailed dunnart and honey possum, suggests that three spectrally distinct cone types are a feature spanning the marsupials.

Modelling oil droplet absorption spectra and spectral sensitivities of bird cone photoreceptors

Birds have four spectrally distinct types of single cones that they use for colour vision. It is often desirable to be able to model the spectral sensitivities of the different cone types, which vary considerably between species. However, although there are several mathematical models available for describing the spectral absorption of visual pigments, there is no model describing the spectral absorption of the coloured oil droplets found in three of the four single cone types. In this paper, we describe such a model and illustrate its use in estimating the spectral sensitivities of single cones. Furthermore, we show that the spectral locations of the wavelengths of maximum absorbance (lambda(max)) of the short- (SWS), medium- (MWS) and long- (LWS) wavelength-sensitive visual pigments and the cut-off wavelengths (lambda(cut)) of their respective C-, Y- and R-type oil droplets can be predicted from the lambda(max) of the ultraviolet- (UVS)/violet- ( VS) sensitive visual pigment.

Tolerance of endolithic algae to elevated temperature and light in the coral Montipora monasteriata from the southern Great Barrier Reef

Photosynthetic endolithic algae and cyanobacteria live within the skeletons of many scleractinians. Under normal conditions, less than 5% of the photosynthetically active radiation (PAR) reaches the green endolithic algae because of the absorbance of light by the endosymbiotic dinoflagellates and the carbonate skeleton. When corals bleach (loose dinoflagellate symbionts), however, the tissue of the corals become highly transparent and photosynthetic microendoliths may be exposed to high levels of both thermal and solar stress. This study explores the consequence of these combined stresses on the phototrophic endoliths inhabiting the skeleton of Montipora monasteriata, growing at Heron Island, on the southern Great Barrier Reef. Endoliths that were exposed to sun after tissue removal were by far more susceptible to thermal photoinhibition and photo-damage than endoliths under coral tissue that contained high concentrations of brown dinoflagellate symbionts. While temperature or light alone did not result in decreased photosynthetic efficiency of the endoliths, combined thermal and solar stress caused a major decrease and delayed recovery. Endoliths protected under intact tissue recovered rapidly and photoacclimated soon after exposure to elevated sea temperatures. Endoliths under naturally occurring bleached tissue of M. monasteriata colonies (bleaching event in March 2004 at Heron Island) acclimated to increased irradiance as the brown symbionts disappeared. We suggest that two major factors determine the outcome of thermal bleaching to the endolith community. The first is the microhabitat and light levels under which a coral grows, and the second is the susceptibility of the coral-dinoflagellates symbiosis to thermal stress. More resistant corals may take longer to bleach allowing endoliths time to acclimate to a new light environment. This in turn may have implications for coral survival.

Determination of chromophore charge states in the low pH color transition of the fluorescent protein Rtms5(H146S) via time-dependent DFT

The red fluorescent protein Rtms5H146S displays a transition from blue (absorbance λmax 590 nm) to yellow (absorbance λmax not, vert, similar453 nm) upon titration to low pH. The pKa of the reaction depends on the concentration of halide, offering promise for new expressible halide sensors. The protonation state involved in the low pH form of the chromophore remains, however, ambiguous. We report calculated excitation energies of different protonation states of an RFP chromophore model. These suggest that the relevant titration site is the phenoxy moiety of the chromophore, and the relevant base and conjugate acid are anionic and neutral chromophore species, respectively.