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Exploring Gradients of Halogens and Zinc in the Surface and Subsurface of Nereis Jaws Rashda K. Khan,† Peter K. Stoimenov,† Thomas E. Mates,‡ J. Herbert Waite,*,†,§ and Galen D. Stucky*,†,| Department of Chemistry and Biochemistry, Materials Research Laboratory, Department of Molecular, Cellular, and DeVelopmental Biology, and Materials Department, UniVersity of California, Santa Barbara, California 93106 ReceiVed April 15, 2006. In Final Form: June 21, 2006 The outstanding mechanical properties of impact-bearing tissues, such as Nereis jaws, make their morphology and chemical composition a subject of particular interest. The complex structure of the jaw was recently reported to exhibit molecular gradients that were closely correlated with stiffness and hardness.18 Accordingly, we have explored the spatial distribution and bonding chemistries of Zn and the halogens in the surface structure of the jaws. Using secondary ion mass spectrometry (SIMS) and scanning electron microscopy (SEM), we found that Cl, Br, and I distributions are enhanced in surface layers of the basal protected portion of the jaw but are shifted to greater depths toward the exposed jaw tip. There are thus two complementary halogen gradients in the jaw: one on the surface that decreases from the base to the tip, coupled to an increasing one in the subsurface layers. The outer surface coating appeared to have granular morphology, in contrast to the anisotropic, fibrous core that dominates the subarchitecture. Using X-ray photoelectron spectroscopy (XPS), we discovered that Zn, I, and Br in the jaws have single chemical environments whereas chlorine is present in two distinct modes (Cl-Zn and Cl-C). Given the inverse relationship between surface exposure and halogen abundance in the jaws, it is unlikely that the halogens contribute directly to mechanical properties such as wear and hardness.
Introduction In biological systems, zinc is frequently found coordinated to imidazolate and thiolate ligands such as histidine and cysteine, functioning either structurally (zinc finger proteins) or catalytically (carbonic anhydrase).1 Significant zinc concentrations are also observed in hard impact-bearing tissues such as mandibles and fangs of insects2,3 and marine invertebrates.4-6 However, the solid-state structure and ligand chemistry of the zinc in these structures remain to be fully explored. It is of further interest that some zinc-containing hard tissues also have significant halogen content.2,4-6 Although the function of halogens in these structures is not well understood, halogens play prominent roles elsewhere in nature. Iodinated phenoxy rings derived from tyrosine molecules make up the thyroid hormone, thyroxine,7 which regulates the basal metabolic rate. Over 3800 organohalogen (Cl, Br, I, and F) compounds, many being investigated as new medicinal drugs, are known natural products from marine and terrestrial organisms.8 Insects, marine algae, sponges, snails, crabs, * Corresponding authors. E-mail:
[email protected]; waite@ lifesci.ucsb.edu. † Department of Chemistry and Biochemistry. ‡ Materials Research Laboratory. § Department of Molecular, Cellular, and Developmental Biology. | Materials Department. (1) Lippard, S. J.; Berg, J. M. Principles of Bioinorganic Chemistry; University Science Books: Mill Valley, CA, 1994. (2) Hillerton, J. E.; Robertson, B.; Vincent, J. F. V. J. Stored Prod. Res. 1984, 20, 133-137. (3) Schofield, R. M. S.; Nesson, M. H.; Richardson, K. A. Naturwissenschaften 2002, 89, 579-583. (4) Perry, C. C.; Grime, G. W.; Watt, F. Nucl. Instrum. Methods Phys. Res. 1988, B30, 367-371. (5) Bryan, G. W.; Gibbs, P. E. J. Mar. Biol. Assoc. U.K. 1980, 60, 641-654. (6) Lichtenegger, H. C.; Scho¨berl, T.; Ruokolainen, J. T.; Cross, J. O.; Heald, S. M.; Birkedal, H.; Waite, J. H.; Stucky, G. D. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9144-9149. (7) Greenstein, J. P.; Winitz, M. Chemistry of the Amino Acids;; John Wiley & Sons: New York, 1961; Vol. 3, Chapter 38. (8) Gribble G. W. Chemosphere 2003, 52, 289-297.
and corals use haloperoxidases to make well-known natural products,9,10 including halogenated amino acids, that are proposed to function in chemical defense8,9,11 and protein sclerotization.12-16 Previously, we have suggested that zinc coordination in Nereis jaws resembles Zn(NHis)3Cl, which defines the coordination chemistry of the Zn insulin hexamer.6 Zinc and histidine concentration gradients reach their highest levels toward the jaw tip,5,6 where stiffness and hardness also peak.18 Elemental distribution maps show that zinc and chlorine are concentrated in the jaw core whereas Br and I occur toward the periphery,6,17 where they appear to be associated with a variety of halogenated amino acids from whole Nereis jaws.17 Whereas this previous work has suggested that inorganic elements differ in their jaw distribution,5-6,17 more detailed studies are necessary to resolve the relationship between the ultrastructure, zinc, the halogen spatial distribution, and the chemical nature. Here we present the results of such a study on the distribution and element environments of Zn and the halogens in Nereis worm jaws. We specifically addressed varying distributions of the halogens and zinc on the top 10 µm of the jaw surface, which was correlated with jaw ultrastructure and elemental bonding environments. Using secondary ion mass spectrometry (SIMS), scanning electron microscopy (SEM), and X-ray photoelectron (9) Butler, A.; Walker, J. V. Chem. ReV. 1993, 93, 1937-1944. (10) Walker, J. V.; Morey, M.; Carlsson, H.; Davidson, A.; Stucky, G. D.; Butler, A. J. Am. Chem. Soc. 1997, 119, 6921-6922. (11) Gribble, G. W. Chem. Soc. ReV. 1999, 28, 335-346. (12) Goldberg, W. M.; Hopkins, T. L.; Holl, S. M.; Schaefer, J.; Kramer, K. J.; Morgan, T. D.; Kim, K. Comp. Biochem. Physiol. 1994, 107B, 633-643. (13) Andersen, S. O. Acta Chem. Scand. 1972, 26, 3097-3100. (14) Hunt, S.; Breuer, S. W. Biochim. Biophys. Acta 1971, 252, 401-404. (15) Welinder, B. S.; Roepstorff, P.; Andersen, S. O. Comp. Biochem. Phys. 1976, 53B, 529-533. (16) Welinder, B. S. Biochim. Biophys. Acta 1972, 279, 491-497. (17) Birkedal, H.; Khan R. K.; Slack, N.; Broomell, C. C.; Lichtenegger, H. C.; Zok, F. W.; Stucky, G. D.; Waite, J. H. ChemBioChem, in press, 2006. (18) Waite, J. H.; Lichtenegger, H. C.; Stucky, G. D.; Hansma, P. Biochemistry 2004 43, 7653-7662.
10.1021/la061027k CCC: $33.50 © 2006 American Chemical Society Published on Web 08/04/2006
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Figure 1. Surface morphology and halogen depth profiles of jaw tip, mid, and base. (a) Whole jaw photograph. Tip to base is a gradient from dark brown to off-white. For SIMS analysis, the jaw was cut into three sections as indicated by black lines through the photograph. SEM of the jaw showed the location of surface analysis and post-SIMS craters on the jaw (b-d). Post-SIMS crater appearance of jaw base and morphology observed with high-resolution SEM (d i-iii). SIMS measured depth profiles of 35Cl, 79Br, and 127I for approximately 10 µm in the jaw surface (e-g).
spectroscopy (XPS) we show that Cl, Br, and I distributions are enhanced in surface layers of the basal portion of the jaw but are more prominent at subsurface layers near the jaw tip. The surface coating appears to be granular in all portions of the jaw, in contrast to the anisotropic, fibrous structure that dominates the interior. Experimental Section Materials. Live Nereis Virens were obtained from the Harbor Bait Company (Edgcomb, Maine). They were decapitated upon arrival, and the heads were stored at -80 °C until use. Jaws were dissected from freshly thawed worm heads, washed with Nanopure water, and air dried. 3-Chloro-L-tyrosine, 3,5-dibromo-L-tyrosine, 3-iodo-L-tyrosine, L-histidine, zinc iodide, and zinc oxide were supplied by Sigma-Aldrich. Potassium periodate, potassium bromide, potassium iodide, and sodium chloride were purchased from Fisher Scientific Company. Zinc chloride was purchased from EM Science. All chemicals were used without further purification. Scanning Electron Microscopy (SEM). An FEI Sirion XL 40 SEM was operated at 3 kV. All jaw samples were gold-sputtercoated prior to imaging. Secondary Ion Mass Spectrometry (SIMS). Nereis Virens jaws were dissected, washed, and air dried as indicated above. The jaw was sectioned with an ethanol-cleaned razor blade into three pieces (tip, mid, and base), shown in Figure 1a-d. SIMS analysis was performed using a 6650 DSIMS (Physical Electronics USA, Chanhassen, MN). An 8 kV O2+ beam at 57 nA was focused to a diameter of approximately 15 µm and rastered in a 100 × 130 µm2 pattern. SEM was used to calibrate the SIMS depth scale. SIMS was performed toward the bulging19 area of the jaw pieces. A low-energy electron flood was used for charge compensation, but because some signal drift due to sample charging was unavoidable, all signals were normalized to 12C (Figure 1e-g). (19) Birkedal, H.; Broomell, C. C.; Khan, R. K.; Slack, N.; Lichtenegger, H. C.; Zok, F. W.; Stucky, G. D.; Waite, J. H. Mater. Res. Soc. Symp. Proc. 2005, 874, L2.8.1/K2.8.1.
Figure 2. Fractured jaw-tip SEM showing two distinct morphologies: fibrous core and nonfibrillar surface coating. The fibers appear to run along the jaw axis. The nonfibrillar surface coating was observed with higher resolution and was shown to vary between 3 and 8 µm in thickness, depending on jaw location (inset a and b). X-ray Photoelectron Spectroscopy (XPS). Jaws were ground under liquid nitrogen with a mortar and pestle. Approximately 2 mg of jaw powder was pressed under a hydraulic press at 5000 psi into flat tablets on double-sided adhesive copper tape. All standard powdered samples were prepared in the same manner as jaw powder, except for hygroscopic solids that were transferred to double-sided adhesive copper tape in a glovebox. The sample area examined by the X-ray was approximately 300 × 700 µm2. High-resolution and survey scans were collected on a Kratos Axis Ultra spectrometer (Kratos Analytical, Manchester, UK). Monochromated Al KR1,2 1486.6 eV radiation was used as an excitation source, using a lowenergy electron flood for charge compensation. Zn 2p, Cl 2p, Br 3d, and I 3d high-resolution spectra were collected at 40 eV pass energy and a 0.1 eV channel width. C 1s and N 1s spectra were collected at 20 eV pass energy. Survey scans were performed using 160 eV pass energy and 0.5 eV channel width. Several regions on ground-
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Figure 3. Cl 2p XPS of pulverized whole Nereis jaws (bottom spectra) were compared to Cl-containing compounds (a). Jaw Cl 2p showed two chemical environments (Zn-Cl, 2p3/2 198.5 eV and C-Cl, 2p3/2 200.7 eV) with higher metal chloride concentration. Zn 2p XPS of pulverized Nereis jaws (bottom spectra) compared to Zn-containing compounds (b). Table 1. Elements Observed on the Surface of Pulverized Whole Jaws element
surface composition, atom %
C N O Cl Br I Zn
67 12 19 0.5 0.6 0.2 0.3
jaw tablets were scanned for consistency. All spectra were accumulated quickly and in fresh areas of powdered samples to avoid artifacts due to beam damage. All binding energies (BE) were referenced to aliphatic C 1s at 284.9 eV.
Results and Discussion A photograph of a whole Nereis jaw (Figure 1a) shows the intricate jaw structure and curvature toward the tip. A color gradient is noted from dark brown (distal tip) to offwhite (proximal base). Presumably, the darker color originates from increased sclerotization and protein cross linking in the direction of the tip. Protein sclerotization in black coral skeletons has been proposed to cause increased pigmentation, hardness, and hydrophobicity.12
Chemical and Ultrastructural Variations with SIMS and SEM. To minimize surface curvature in test specimens, we sectioned a jaw into three portions: tip (Figure 1b), mid (Figure 1c), and base (Figure 1d). Oxygen beam craters post-SIMS were observed with SEM (marked with white arrows in Figure 1b1d). The corresponding halogen depth profiles of the three jaw sections measured 35Cl, 79Br, and 127I (Figure 1e-g). The changing relationship between the preponderance of halogen distribution and sampling location in each jaw shows both longitudinal (baseto-tip) and transverse (surface-to-core) chemical gradients. Longitudinally, halogens undergo a decreasing concentration gradient and are sequestered on the outer surface micrometer in the base. This splits into surface and subsurface distributions in the midsection, and in the tip, the surface distributions have completely given way to those in the subsurface. Depth profiles also revealed that the three halogens have only partially overlapping distributions at any given sampling location. The jaw tip (Figure 1e) showed 127I, for example, concentrated closest to the jaw surface, peaking from 2.5 to 4 µm with the signal practically disappearing by the time that the 10 µm depth is reached. 79Br counts peaked from 3 to 6 µm and fell slightly at 10 µm into the jaw surface. 35Cl intensity, in contrast, was observed to plateau from 4 to 10 µm, which may be attributed to the higher Cl concentration in the interior of the jaw tip.6 Thus, halogens
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Figure 4. I 3d XPS of pulverized Nereis jaws (bottom spectra) compared to that of I-containing compounds (a). Br 3d XPS of pulverized Nereis jaws (bottom spectra) compared to that of Br-containing compounds (b).
are not concentrated together on the jaw-tip surface, iodine being more prevalent near the surface, with bromine and finally chlorine predominating as etching proceeded. Transversely, jaw-tip halogens undergo an increasing concentration gradient from the surface to the core. Mid-jaw halogen counts exhibited a distinct bimodal spatial distribution (Figure 1f); the first ranged from 0 to 1 µm, and the second, from 1 to 8 µm. The bimodality appeared to apply equally to 35Cl, 79Br, and 127I. In both the first and second 127I peaks, they tended to peak closer to the surface. This more complex halogen distribution may be indicative of a transition from halogenated tyrosine-rich proteins (∼0 to 1 µm) to halogenated histidine-rich proteins in the next peak (∼1 to 8 µm). In the base (Figure 1g), 35Cl, 79Br, and 127I were all abundant within the first 1 µm surface layer. Transversely, jaw-base halogens exhibit a decreasing surface-to-core concentration gradient. Zooming in on the jaw-base SIMS crater (Figure 1d i-iii), the interior morphology after beam exposure is observed. In live worms, the base region (off-white region in Figure 1a) is filled with pulp tissue and rooted in the proboscis. It is exposed to the environment only when the jaws are fully everted and hence not subjected to significant loads or impacts as in the jaw tip. From the SIMS experiments, we infer that the bulging side of the jaw base has a 1-µm-thick halogen-rich (and zinc and histidine-deficient) outer layer that may function largely to protect
the interior against bacterial/viral/fungal attack because bromotyrosine derivatives show antifouling properties.20 Ultrastructural variations in the jaw tip were observed and compared with SIMS analyses. In contrast to the base and mid regions, the outermost 1 µm of the tip is depleted of halogens. Here, we observed two structurally distinct morphologies of a fractured jaw tip: a fibrous interior and nonfibrillar surface coating (Figure 2). This surface coating ranged from ∼3 µm (Figure 2, inset a) to ∼8 µm (Figure 2, inset b) in thickness and has a granular appearance. This variable coating thickness is not only observed in the transverse direction but also present in the longitudinal direction, which may be explained by differing demands of wear resistance. However, it is possible for the halogens to be incorporated as components of the nonfibrillar surface coating, but we suspect that they begin closer to the interface of the fibrous interior. The composition and mechanical properties of the top few micrometers of jaw coating remain to be determined. Because Cl abundance is linearly correlated with Zn concentration,6 it is appropriate to ask whether the 35Cl jaw SIMS depth profiles also correlate to zinc and histidine distributions along analyzed jaw surfaces. The jaw-tip zinc and histidine most likely begin at ∼4 µm (depth at which 35Cl commenced) from the surface because zinc and histidine contents are highest in the jaw-tip region.6,18 We suspect that the zinc in the mid-jaw portion (20) Fusetani, N. Nat. Prod. Rep. 2004, 21, 94-104.
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Figure 5. C 1s (a) and N 1s (b) XPS of pulverized Nereis jaws (bottom spectra) compared to those of pristine L-histidine and halogenated tyrosines.
is not present in the first peak (0 to 1 µm) and that the second peak (1 to 8 µm) region may correspond to a smaller quantity of zinc and co-localized His-rich protein. There is very little zinc in the jaw-base region,5,6 so the Cl seen there must reflect mostly chlorinated tyrosines, in agreement with our XPS analyses. Chemical Bonding Environments with XPS. The abundance and differential distributions of the halogens suggested the possibility of a variety of chemical bonding environments. XPS was used to detect the quantity and possibility of multiple chemical bonding environments on the surface of pulverized whole jaws. A wide scan (survey) spectrum showed C, N, O, Cl, Br, I, and Zn; quantitatively, a primarily organic composition was observed (Table 1). Chlorine 2p high-resolution XPS analyses confirmed two distinct valence states: one at 198.5 eV, attributable to Cl-Zn, and the other at 200.7 eV, attributable to Cl-C. For the first time, we obtained an indication of Cl quantities in whole jaws, finding Cl-Zn to account for 60% and Cl-C to account for 40% of the Cl content (Figure 3a). These high-resolution Cl 2p assignments are in agreement with the survey Cl (0.5 atom %) and Zn (0.3 atom %) observed in Table 1. The comparison of XPS data of some model Cl-containing compounds (3-chloro-
L-tyrosine, NaCl, and ZnCl2) suggests similarity between jaw Cl
and the chemical environments of 3-chloro-L-tyrosine and NaCl. Quantitatively, Cl- appears to be the more abundant form of Cl in the jaws (Figure 3a). Existing literature reports alkali chloride BE ranging from 198 to 200 eV,21 which is consistent with our results. Furthermore, the two valence states of Cl are consistent with previous work that revealed a Zn(NHis)3Cl motif 6 and more recently chlorinated tyrosyl residues.17 The Zn 2p3/2 peak in the Nereis jaws was observed to have a BE of 1021.9 eV and suggests a single valence state (Figure 3b). XPS measurements of model compounds ZnO, ZnI2, and ZnCl2 did not suggest a match with the chemical environment of zinc in the jaw. The BE range of Zn 2p in a study of zinc nitride thin filmssZn3N2 (1022.0 eV), ZnO (1021.8 eV), and metallic zinc (1021.7 eV)sare also interesting.22Our results have confirmed a single chemical environment in jaw zinc and suggest (21) Moulder J. F.; Stickle, W. F.; Sobol, P. E.; Bomben, K. D. In Handbook of X-ray Photoelectron Spectroscopy; Chastain, J., King, R. C., Jr., Eds.; Physical Electronics: Eden Prairie, MN, 1995. (22) Fatsuhara, M.; Yoshioka, K.; Takai, O. Thin Solid Films 1998, 322, 274281.
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Scheme 1. Zn Coordination and Covalent Cross Linking in the Nereis Jaw Tipa
a Animation of worm about to strike a victim with a pair of jaws that are exposed only as the proboscis is everted (a). Brominated or iodinated hisitidyl residues are modeled in the Zn(Nδ1His)3Cl motif (b). Cl, Br, and I are bonded to C1 and C2 of tyrosine phenols (c).
a binding energy consistent with the Zn environment in Zn(NHis)3Cl. Because XPS observations of Cl 2p supported the existence of two distinct chemical environments and natural occurrences of halides have been previously reported,23-25 the possibility of multiple valence states in jaw Br and I was also investigated. The Nereis jaws’ Br 3d BE was compared to 3,5-dibromo-L-tyrosine and KBr (Figure 4a). The jaw Br 3d (70.8 eV) spectrum suggests a single valence state, and the BE is closest to that of 3,5-dibromoL-tyrosine (70.5 eV). Subsequently, high-resolution I 3d XPS was performed on jaw powder and compared with 3-iodo-L-tyrosine, ZnI2, KI, and KIO4 (Figure 4b). The I 3d spectrum of the jaws confirmed a single electronic state and a BE identical to that of 3-iodo-Ltyrosine (620.7 eV). This observation, when compared to the jaw Br 3d, may suggest a uniform chemical environment of iodine in the jaws. Both brominated and iodinated tyrosyl and histidyl residues have been characterized from hydrolysates of Nereis jaws.17 Although we are unable to distinguish these two environments with great resolution in the present study, this may be reflected in Br 3d and I 3d XPS binding energies and the comparison of SIMS halogen depth profiles in the jaws. The slight BE shift of the jaws from dibromotyrosine (Figure 4a) and the fact that 79Br follows 35Cl depth profiles more closely (Figure 1e-g) may be due to the presence of more Br-histidyl bonding. In contrast, Nereis jaw I 3d BE precisely matched standard 3-iodo-L-tyrosine BE (Figure 4b), revealing a more uniform chemical environment, and SIMS 127I depth profiles remained unique and always concentrated closer to the surface in all jaw parts that were examined. Why nature chooses to concentrate this particular halogen on the surface is an interesting question for further (23) Kylin, H. Z. Physiol. Chem. 1929, 186, 50-84. (24) Amat, M. A.; Srivastava, L. M. J. Phycol. 1985, 21, 330-333. (25) Manley S. L. J. Phycol. 1983, 19, 118-121.
investigation. Interestingly, with regard to the rates of iodination in aromatic amino acids, it is of great importance to note kinetic studies that show iodinated tyrosine formation is faster than histidine iodination. Indeed, the formation of diiodotyrosine is 100 times faster than diiodohistidine in citrate buffer at pH 6.26 Another study used a protein (ribonuclease A) rich in histidine and tyrosine to show that the iodination of tyrosyl residues is always preferred over that of histidine.27 Thus, we suspect that iodine in the Nereis jaws is largely associated with tyrosine residues. Given that the Nereis jaws are composed mostly of protein, we pursued high-resolution carbon and nitrogen studies with XPS. C 1s environments in Nereis jaws, L-histidine, 3-iodo-Ltyrosine, 3,5-dibromo-L-tyrosine, and 3-chloro-L-tyrosine compounds were compared (Figure 5a). To the best of our knowledge, this is the first report of XPS on naturally occurring halogenated amino acids. We used halogenated model compounds as controls in our studies and showed the C-halogen bond BE at approximately 286.6 eV. The jaw C 1s spectra appear to be more complex than those of simple halogenated amino acid derivatives and L-histidine. Carbon spectra for jaws illustrated five main components: C-C (284.9 eV), C-N/O (285.7 eV), C-O/halogen (286.5 eV), N-CdO (288.2 eV), and O-CdO (289.1 eV). Other examples of halogenated compounds, such as CH2Br2 and C6H5Cl, have a BE of 287.1 eV,21 which further supports the C-halogen bond presence in our samples. C 1s spectra have been reported for several free amino acids, indicating carboxylic carbon (O-CdO),28,29 which is consistent with the model halogenated derivatives that we tested. On the contrary, the presence of a large N-CdO component in the jaws (26) Li, C. H. J. Am. Chem. Soc. 1944, 66, 225-227. (27) Covelli, I.; Wolff, J. J. Biol. Chem. 1966, 241, 4444-4451. (28) Zubavichus, Y.; Zharnikov, M.; Yongjie, Y.; Fuchs, O.; Heske, C.; Umbach, E.; Tzvetkov, G.; Netzer F. P.; Grunze, M. J. Phys. Chem. B 2005, 109, 884-891. (29) Zubavichus, Y.; Zharnikov, M.; Shaporenko, A.; Fuchs, O.; Weinhardt, L.; Heske, C.; Umbach, E.; Denlinger, J. D.; Grunze, M. J. Phys. Chem. A 2004, 108, 4557-4565.
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suggests an amide carbon environment and thus contributions from the protein backbone. Pristine L-histidine was also tested because it accounts for more than a third of the jaw protein. Here, histidine spectra are consistent with those reported previously28 and showed a quantitatively higher component of C-N. N 1s spectra showed a comparison of pristine halogenated tyrosine compounds, L-histidine and Nereis jaws (Figure 5b). Halogenated tyrosines all exhibited BEs between 401.7 and 401.9 eV, representative of protonated amino groups (NH3+). L-Histidine showed four main components:28 NH3+ (401.6 eV), NH2 (400.7 eV), NH (399.9 eV), and tertiary N (398.5 eV), representative of the nitrogen in imidazole and amino groups. The jaw N 1s spectra also showed four components, primarily consisting of the NH moiety at 400.0 eV, which is representative of amide bonds in peptides (protein). Modeling of Zn, Amino Acid Cross Links, and the Nereis Worm. Nereis jaws are mounted at the end of an evertible proboscis and exposed when the animal strikes (Scheme 1a) or burrows through sediment.30 A speculative glimpse of some of the chemical interactions in a jaw tip is illustrated in Scheme 1b and is consistent with the X-ray absorption spectroscopy of the Nereis jaw,6 energy dispersive spectroscopy of halogens,17 the characterization of halogenated amino acids,17 and the halogen gradients established here. In this model, Zn2+ is coordinated to the Nδ1 of three histidine imidazoles and a chloride ion. The imidazole ligands can be either unmodified or with halogenated Cδ2 and C1 (i.e., X ) H, Br, or I), although we suspect mostly bromination (Scheme 1b). C1 and C2 of the tyrosine phenoxy (30) Dorgan, K. M.; Jumars, P. A.; Johnson, B.; Boudreau, B. P.; Landis, E. Nature 2005, 433, 45.
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rings are ring coupled or halogenated with X (X ) Cl, Br, I) (Scheme 1c).
Conclusions In summary, we have obtained high-resolution surface depth profiles of halogens that vary according to location in the jaw. Jaw surfaces showed halogen gradients varying in transverse (surface-to-core) and longitudinal (base-to-tip) directions. The jaw-tip halogen atoms appeared to be depleted in the top 1 µm granular surface coating and occurred as diffuse distributions into the fibrous core. In the mid jaw, a broad bimodal distribution of halogens appeared in surface and subsurface layers, whereas in the jaw base, halogens seemed preferentially sequestered toward the surface. We have found that Zn, I, and Br in the jaws have single chemical environments, whereas chlorine is present in two chemical binding environments. Spatial compositional changes in the surface coating suggest its complex and changing functions in the jaw. At the tip where wear resistance properties are most needed, the halogens are scarce. In contrast, at the base which is compliant, the halogen content is high, and the distribution is narrow. Further elemental, mechanical, surface energy, and microbial analyses of Nereis jaws are underway. Acknowledgment. We thank Peter Allen for the artistic rendition of the Nereis worm. Chris Broomell is acknowledged for helpful discussions. Financial support from NIH BRP DE014672 is gratefully acknowledged. R.K.K. is grateful for support by the University of California Systemwide Biotechnology Research & Education Program GREAT Training Grant 2005-240. This work made use of MRL Central Facilities supported by the MRSEC Program of the National Science Foundation under award no. DMR05-20415. LA061027K
