Water Influences on the Copper Active Site in Hemocyanin - The

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Water Influences on the Copper Active Site in Hemocyanin €nhense,† Daniel Panzer,† Christian Beck,‡ Michaela Hahn,† Jochen Maul,† Gerd Scho ‡ ,§ Heinz Decker, and Emad F. Aziz* †

Institut f€ ur Physik, Johannes Gutenberg-Universit€at, Staudinger Weg 7, 55099 Mainz, Germany, Institut f€ ur Molekulare Biophysik, Johannes Gutenberg-Universit€ at, Welderweg 26, 55099 Mainz, Germany, and § Helmholtz-Zentrum Berlin f€ ur Materialien und Energie, Albert-Einstein-Strasse 15, 12489 Berlin, Germany ‡

ABSTRACT Active metal sites play a key role in the biochemistry of oxygen transport by hemocyanins. Observing the changes in the local electronic structure of the copper sites upon oxygenation is thus essential for understanding their biological functionality. Here, direct access to the electronic structure of the active copper sites in hemocyanin is achieved via L-edge X-ray absorption spectroscopy under physiological conditions. We compare the deoxygenated and the oxygenated states of native hemocyanin and find evidence that the oxygenation does not simply switch the copper valence state between Cu I and Cu II, as assumed classically. In the deoxygenated state, water molecules can enter the active site and keep the copper atoms partially oxidized. The role of water in this process has never been revealed before for lack of L-edge spectroscopy on copper in solution. Besides providing a more detailed electronic picture for the oxygenation process, this study opens a new chapter in investigating the function of proteins under in-vivo-like conditions. SECTION Biophysical Chemistry

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hereas in hemoglobin the dioxygen is bound by an iron atom, in hemocyanin, the oxygen molecules are bound as peroxide between two copper atoms in a side-on coordination (see Figure 1). Hemocyanins are widespread metalloproteins occurring freely dissolved in the hemolymph of arthropods and mollusks and bind up to 160 oxygen molecules in a cooperative manner at their active sites.1-5 Molluskan hemocyanins form hollow cylinders composed of large subunits folded into 7-8 functional units with an oxygen binding center each, and arthropod hemocyanins occur as multiples of hexamers, depending on the species.1-8 Despite these structural differences, hemocyanins show very similar active sites, as do all proteins belonging to the type-3 copper protein family.1 Dioxygen binding causes a change in the electronic structure of the copper active site accompanied by a change in the valence and the coordination chemistry.6,7,9,10 This change is transferred to the protein matrix via two sets of three histidines, each set coordinating one of the two copper atoms.5,7,10 These changes are the origin of the cooperative conformational change between the subunits, as confirmed by various biophysical methods.11,12 However, the electronic interaction between the copper atoms, the oxygen molecule, and the amino acids at the active site is still under debate.13,14 X-ray absorption spectroscopy (XAS) is an ideal tool for probing the electronic and geometric structure of the active site. Hard X-ray absorption spectroscopy studies have been carried out at the K-edge of copper and considerably

r 2010 American Chemical Society

contributed to clarifying the complex local geometric structure of the hemocyanin active site.14-17 However, these studies only briefly touch upon the subject of oxidation states, which are determined by the d band occupation. For the purpose of investigating the particularities of the oxidation/ valence changes of first-row (3d) transition metals, soft X-ray spectroscopy at the L2;3 edge (2p1/2;3/2 f 3d transitions) is obviously more suitable. In principle, similar information is available from, for example, the pre-edge structure of the K-edge, but for late transition metals like copper, its intensity is small. For L-edge spectra, the dipole transition probability is high, and the longer intrinsic core hole lifetime of p orbitals results in sharper spectral features than those at the K-edge. Furthermore, L2;3-edge features are directly proportional to the amount of d character of unoccupied valence orbitals of the metal. This partly applies to Cu I as well, as the tendency of copper toward covalence often causes noninteger values for the number of d electrons. Therefore, even with a nominally full d shell, dipole transitions are not completely impossible. In fact, all coordination chemistry involves the d orbitals of the transition metal, and monitoring the changes in these orbitals delivers direct information about the alteration of the local electronic structure of the probed element.18 This is especially useful where coordination with changing ligands is possible, Received Date: March 10, 2010 Accepted Date: May 3, 2010 Published on Web Date: May 06, 2010

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DOI: 10.1021/jz100313q |J. Phys. Chem. Lett. 2010, 1, 1642–1647

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Figure 2. Cu L3 XAS spectra of hemocyanin in solution in the deoxygenated (black) and the oxygenated state (red).

both a white line at 932 eV and a broad peak around 936 eV. These latter contributions to the copper spectrum are characteristic for monovalent copper, where the d shell is fully occupied24 and will be enhanced by MLCT transitions.19 If we assume the classical picture of the deoxy form of hemocyanin correlated to an exclusive Cu I state,13 we would expect neither the preceding white line that indicates the presence of divalent copper nor MLCT transitions (due to the lack of a suitable ligand). Accordingly, there are two possible scenarios; one is the presence of a mixture of the classical deoxy form with the oxy form (due to an inefficient deoxygenation process) or a met form (possibly by photo-oxidation or a chemical reaction). Another possibility is that this classical picture with a pure Cu I state upon dioxygen release is not a valid interpretation. In order to test the first case, we used two different methods to deoxygenate the hemocyanin. First, we exposed the protein solution to an oxygen-free atmosphere (99.999% helium medium; technical details in the Supporting Information), which removes the bound dioxygen over time. Second, we added sodium dithionite to the solution to deoxygenate the protein instantly by irreversibly binding all oxygen molecules to the dithionite. A comparison of the spectra obtained from the two methods is shown in Figure 3a. Both methods of deoxygenation yield almost identical spectra. This addresses two important points; the occurrence of any remnants of oxygenated hemocyanin in the helium-deoxygenated solution can be ruled out, and at the same time, the deoxygenation with sodium dithionite does not seem to damage the protein at the active site. For further confirmation, the sample was kept under helium flow for several hours, and we repeatedly measured the spectra of the copper L-edges during this time, as shown in Figure 3b. It is quite clear that the protein lost the dioxygen, and no traces of the oxy form remained as the spectra did not change as a function of exposure time to helium. Actually, such repeated measurements also rule out beam damage (specifically photo-oxidation of the active site). As determined previously from K-edge measurements,16 beam damage to the protein, or at least the active site, appears

Figure 1. Schematic drawing of the hemocyanin active site in its two classic states. Each copper atom is complexed by a set of three histidines, which form the connection to the protein backbone. (a) Deoxygenated state without any ligands. (b) Oxygenated state with oxygen bound as peroxide in a side-on coordination.

for example, to monitor metal-to-ligand charge transfer (MLCT) as well as ligand-to-metal charge transfer (LMCT) transitions.19 Nevertheless, the implementation of soft XAS of high vapor pressure solutions was hampered for a long time because of the need for a high vacuum. However, in the past few years, these problems have been overcome by the use of high-speed liquid jets or flow cells equipped with soft X-ray-transparent ∼100 nm-thick silicon nitride membrane windows, opening the way to study chemical systems in solution.20,21 We recently extended these studies to proteins in physiological solutions and recorded the Fe L-edge spectra of hemoglobin versus hemin18 and of the catalase active center.22 Figure 2 shows how oxygenation of the hemocyanin molecules impacts the spectral signature on the L-edge. For oxygenated hemocyanin only a single peak (white line) at 932 eV with a small shoulder toward lower energies is visible, corresponding to the transition of a 2p3/2 electron into the empty 3d state.23-25 The main peak at 932 eV is in agreement with the expected copper valence state of Cu II in oxygenated hemocyanin. The appearance of the shoulder suggests an unequal electronic structure of the copper atom pair introducing two white line peaks. This can be explained by the slightly asymmetrical binding of the oxygen molecule between the two copper atoms in hemocyanins or by the differing arrangements of the three histidines around each of the two copper atoms, as previously shown by X-ray crystallography for different hemocyanins.7,8 The spectrum of the deoxygenated form displays entirely unexpected features. It exhibits


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probes the local electronic structure of the d orbitals and reflects clearly any changes that take place in these orbitals via any molecular counterpart. This however raises the question why the presence of water was not detected sooner. EXAFS measurements in particular should have been able to locate water molecules in close proximity to the copper atoms of an active site, even though the interpretation of the EXAFS data pertaining to hemocyanin has changed quite a bit over the last decades. Some early EXAFS studies commit to two neighboring atoms (the nitrogen atoms of two histidines) for the deoxy state and four neighboring atoms for the oxy state (with oxygen in an end-on coordination).15-17 A newer study increases these numbers to three to four neighboring atoms for deoxy and five to six neighboring atoms for oxy in the generally accepted side-on configuration.14 Still, there is a consensus that the number of neighboring atoms decreases by two upon deoxygenation, which would not be the case with water as a ligand. Earlier studies were somewhat different in respect to water content or physical state (lyophilized powder,26 a gel,15 frozen15,17 or embedded in a sucrose matrix14 designed to retain the protein in its native state), but at least the latter two methods should have been able to detect this irregularity. Then, there is the fact that the deoxygenated state of hemocyanin is generally more tolerant toward modification (in structure and behavior) as there is no rigid default for realizing the oxygen-free state; therefore, the affinity for water may differ for other hemocyanins. However, again, the only EXAFS study comparing different hemocyanin species14 confirms this general trend for the deoxy forms but offers no indication of an aberration specific to Homarus americanus; therefore, there is no conclusive explanation at this time. Since a XAS measurement is inherently a superposition of the response of all active sites within the area exposed to the beam, it is difficult to specify the distribution of water molecules to the copper atoms. In Figure 4, we show two geometries representing the two main options, a bridging and a nonbridging configuration. The nonbridging variant would be less rigid and fit better with the increased copper distance observed in deoxygenated hemocyanin. Still, the exact number of water ligands per copper atom is unknown at this time and may even differ between zero and two for different active sites. To shed more light onto the change of the electronic structure of hemocyanin upon binding or release of dioxygen, we measured spectra of copper ions dissolved in aqueous solution as well as copper complexes with imidazole or histidine. Initially, an aqueous CuCl2 solution at 1000 mM concentration was measured. The spectrum in Figure 5a exhibits the characteristic white lines at the L3- and L2-edges at 931.3 and 951.6 eV, respectively. Upon dilution to 5 mM, we observe substantial changes in the spectra. The feature related to the MLCT is enhanced, which could be induced by the absence of the Cl- counterion in direct as well as indirect interaction with copper ions. This will lead to mixing of the states between the metal d orbital and the water p orbitals.27 Adding imidazole (25 mM CuCl2 þ 100 mM imidazole, as shown in Figure 5c), which complexes with the copper via a nitrogen atom,7,10

Figure 3. Comparison of deoxygenation methods and long-term stability. (a) L3,2-edge XAS spectra of hemocyanin solution deoxygenated by means of exposure to a helium atmosphere for several hours (red graph) and by adding sodium dithionite to the protein solution (black graph), respectively. (b) Cu L3,2 XAS spectra recorded consecutively (starting with the topmost one) over a time of 2 h and 20 min from a deoxygenated hemocyanin.

to be minimal and can hence be neglected, at least in cases like ours with relatively low intensity due to a small exit slit and attenuation by the membrane and the helium atmosphere. In the second scenario the deoxy form of hemocyanin does not represent an entirely monovalent state. Since water molecules are the predominant molecules in solution when no oxygen is present, it is most likely that the d orbitals of copper hybridize with the LUMOs of neighboring water molecules. The result is a MLCT from the copper center to the water molecules, which mimics the active site being partially oxidized. This also allows a 2p-3d transition, which accounts for the presence of the peak at 932 eV.19 The peroxide bound in the oxy state has no vacant orbitals for accepting MLCT, and therefore, the oxygenated hemocyanin exhibits no such transition. There is currently no crystal structure available for Homarus americanus hemocyanin, but as mentioned before, the active site is highly conserved in all type-3 copper proteins. Therefore, it is fair to discuss some structural aspects based on the similar structure of Limulus polyphemus hemocyanin, which is also an arthropod hemocyanin.8 Here, dozens of water molecules are present, some of them located in close proximity to the active-site copper atoms. The high flexibility of the protein matrix in solution at room temperature allows water molecules to enter the active site, where they can interact with the copper atoms when no dioxygen molecule is present. Ligands like O2 and H2O complex with the transition metal via the d orbitals. The applied L-edge XAS directly


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Figure 4. Schematic (2D) drawings of two proposed bond geometries for the deoxy state with water molecules as ligands. (a) This scheme shows a configuration somewhat similar to that of oxy hemocyanin with two water molecules instead of peroxide bridging the center of the site. (b) Here, the copper atoms have access to two water molecules each.

Figure 5. L2,3 XAS spectra of copper in different aqueous solutions, (a) 1000 mM Cu(II)Cl2, (b) 5 mM Cu(II)Cl2 (dotted line shows deoxy hemocyanin for comparison), (c) 25 mM Cu(II)Cl2 þ 100 mM imidazole, and (d) 50 mM Cu(II)Cl2 þ 200 mM histidine. White line contributions to the spectra are shaded red, and contributions from other transitions are shaded blue (L3-edge only).

causes an increase in the relative white line intensity. Imidazole is the first neighbor of the copper in the protein; nevertheless, in hemocyanin, imidazole will not be in such a strong interaction with the copper active site as in a simple solution of Cu II and imidazole. Adding histidine (50 mM CuCl2 and 200 mM histidine at pH 5, as shown in Figure 5d) increases the peak/step at 935 eV relative to the white line intensity. The copper ions in this case are chelated by the carboxylate group of the amino acids and tend to be in a more Cu I like state. Diluting the mixture CuCl2 þ histidine (see Supporting Information Figure S2) results in an increasing prevalence for the Cu I state as well. These model experiments allow tracking of the effect of charge transfer on the L-edge of copper. The same test on dissolved Cu I failed because it disproportionated too quickly into Cu II and Cu 0. In addition to revealing the change of the local electronic structure for the copper in the active site of hemocyanin upon oxygenation, we have shown a clear electronic fingerprint for the role of water in this process. Water can replace the dioxygen upon deoxygenation, attacking the active site of the hemocyanin and withdrawing electrons from the d orbital of the copper. This is shown here by direct access to the d orbital via L-edge XAS under physiological conditions. Accordingly, the role of water complements our classical understanding of the oxygenation process. The copper atoms at the affected sites remain partially oxidized and do not simply switch between Cu I to Cu II. In one proposed deoxygenated


structure, the water molecules close to the copper site partially mimic the role of the dioxygen in the oxygenated form; the other would probably assist the spatial separation of the copper atoms in the deoxygenated state. At this time, we are unable to determine the purpose of this interaction, and therefore, we can only speculate whether it is vital to the oxygen exchange or if only some hemocyanin types are affected similar to, for example, induced tyrosinase activity. Regarding biological functionality of metalloproteins, this study opens the door to investigating other metalloproteins and their interaction with water molecules using XAS at the L-edge under physiological conditions.

EXPERIMENTAL SECTION X-ray Spectroscopy. The XAS measurements of the copper L3/2-edge were performed at the U41-PGM undulator beamline at HZB/BESSY, Berlin. The setup used for all measurements was the end-station Liquidrom, previously described elsewhere.28 In addition to the standard flow-cell setup where the flowing liquid is separated from the vacuum by a silicon nitride membrane, an open static cell was used to allow for a better signal-to-noise ratio. In this case, the main chamber is separated from the differential pumping stage by a similar silicon nitride membrane and flooded with helium. Inside of the main chamber, the primary beam hits the sample solution through a slit at the side of the cell. In both setups, the emitted

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X-ray fluorescence is detected by a semiconductor diode positioned at a slight angle. Sample Preparation. We used hemocyanin from the American lobster Homarus americanus. The hemolymph was obtained by puncturing of the animal and purified via ultracentrifugation. The concentration was adjusted to 60 mg/mL, and the quality was checked by UV/vis spectroscopy, as detailed in the Supporting Information.

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SUPPORTING INFORMATION AVAILABLE An extended description of sample preparation, XA spectra for varying concentrations of Cu(II)Cl2 and histidine, and a comment on the radiation dose. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION Corresponding Author: *To whom correspondence should be addressed. E-mail: Emad. [email protected].

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ACKNOWLEDGMENT We thank Bernd Petereit and Stefanie Greil

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for beamtime assistance. This work was granted by the HelmholtzGemeinschaft via the young investigator fund VH-NG-635, the DFG (H. D., G.S.) and the Center for Computational Sciences in Mainz (H.D.). (17)

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Water Influences on the Copper Active Site in Hemocyanin - The

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