Time-Resolved Spectroscopic Studies of Radiationless Decay

Nicole Haralampus-Grynaviski, Carla Ransom, Tong Ye, Małgorzata Rôżanowska, Marta Wrona, Tadeusz Sarna, and John D. Simon. Journal of the American ...
1 downloads 0 Views 69KB Size
1478

J. Phys. Chem. B 2001, 105, 1478-1483

Time-Resolved Spectroscopic Studies of Radiationless Decay Processes in Photoexcited Hemocyanins James S. Floyd, Nicole Haralampus-Grynaviski, Tong Ye, Bo Zheng, and John D. Simon Department of Chemistry, Duke UniVersity, Durham, North Carolina 27708

Maurice D. Edington* Department of Chemistry, Florida A&M UniVersity, Tallahassee, Florida 32307 ReceiVed: August 30, 2000; In Final Form: December 12, 2000

Photoacoustic calorimetry and femtosecond transient absorption spectroscopic techniques have been used to study the electronic relaxation and ligand binding reactions that occur following photoexcitation of the 345 nm peroxide-to-copper ligand-to-metal charge transfer (LMCT) band of oxy hemocyanin (Hc) from Limulus polyphemus and Busycon canaliculatum. The results indicate that the LMCT state in both proteins decays to the ground state via a nonradiative mechanism with a time constant of ∼610 fs. The short lifetime of the LMCT state measured in this work provides an explanation for why the oxy Hc excited state exhibits a low fluorescence yield. The results also suggest that photoexcitation of oxy Hc leads to the dissociation of O2 (by a thermal or photochemical process) and that the O2 molecule subsequently recombines with the metal center on the 80 ps time scale. The time-resolved data reveal that the dynamics in the vicinity of the active site are identical for the two types of Hcs studied, which is consistent with the similarity of the active sites in various Hcs.

Introduction Hemocyanins (Hcs) are large multisubunit copper-containing proteins that function as the dioxygen carriers for a wide variety of arthropods and mollusks. Hcs from both types of organisms have similar active-site electronic structures, but their quaternary structures differ significantly.1-3 Molluskan Hcs are composed of 350-400 kDa subunits that are each comprised of 7-8 covalently linked 50 kDa functional units. Each functional unit contains one binuclear copper site and consists of ∼400 amino acid residues; the functional units assemble as decamers or didecamers in the blood. Arthropod Hcs exist as hexamers or multple hexamers of 75 kDa subunits. Each subunit contains ∼650 amino acid residues and a single binuclear copper site. X-ray crystallographic studies performed on Hcs from various arthropods and mollusks show that the active-site geometries of both types of proteins are highly conserved.4-7 In deoxygenated (deoxy) Hc from the arthropod Limulus polyphemus, each Cu(I) atom is coordinated to three histidine ligands in a slightly distorted trigonal planar geometry with a Cu-Cu distance of 4.6 Å.7 When dioxygen binds to the active site, the two Cu(I) atoms are oxidized to Cu(II), and two electrons are transferred to dioxygen, forming peroxide (O22-). In oxygenated (oxy) Hc from L. polyphemus5 and Octopus dofleini4 (mollusk), each Cu(II) atom is pentacoordinated, and the Cu-Cu distance is ∼3.6 Å. The oxy and deoxy Hc active-site structures also exhibit significant differences in the lengths and orientations of the Cu-histidine bonds. The results of numerous theoretical and experimental investigations performed on Hcs and model compounds have led to a detailed understanding of the electronic structure of the oxy Hc metal center.8-14 Dioxygen binds symmetrically to both * Corresponding author. E-mail: [email protected].

copper atoms in a side-on (µ-η2:η2) bridging configuration, as depicted in Figure 1.15 Significant overlap between the peroxide π orbitals and copper d orbitals results in the appearance of two absorption bands in the oxy Hc spectrum that are absent in the deoxy Hc spectrum. These bands correspond to ligand-tometal charge-transfer (LMCT) transitions and are centered at 345 nm (O2-2 π*σ f Cu(II) dx2-y2) and 570 nm (O2-2 π*v f Cu(II) dx2-y2), as shown in Figure 2.16 Despite the wealth of information known about the structural and chemical properties of various Hcs, the excited-state photophysics of the oxy Hc metal center and the time scales and mechanisms of the dioxygen binding and dissociation reactions have not been well characterized. For instance, it is unclear why the LMCT state of oxy Hc does not exhibit any observable room-temperature fluorescence emission, how the active-site chemistry is mediated by the surrounding protein environment, and if the dioxygen binding and dissociation mechanisms differ for arthropod and molluskan Hcs. Understanding how the protein environment influences the chemical and spectroscopic properties of the Hc metal center is an important issue in the field of bioinorganic chemistry because of the increasing interest in using the Hc active-site structure as a model for developing synthetic copper compounds that bind dioxygen for activation and transport reactions.17-21 Experimental investigations of hemoglobin (Hb) and myoglobin (Mb) using transient absorption and laser photolysis methods have contributed greatly to the understanding of the molecular mechanism of reversible ligand binding to the heme centers of those systems.22-28 It seems reasonable to expect that similar studies would yield useful information on the structural and functional properties of Hcs as well. In the present study, femtosecond transient absorption and photoacoustic calorimetry techniques are used to characterize the relaxation (electronic

10.1021/jp003123e CCC: $20.00 © 2001 American Chemical Society Published on Web 01/25/2001

Radiationless Decay in Photoexcited Hemocyanins

Figure 1. Structure of the binuclear copper site of oxy hemocyanin.

Figure 2. Absorption spectrum of oxy hemocyanin. The bands centered at 345 and 570 nm correspond to the O2-2 π*σ f Cu(II) dx2-y2 and O2-2 π*σ f Cu(II) dx2-y2 ligand-to-metal charge-transfer transitions, respectively.

and vibrational) and ligand recombination dynamics that occur following photoexcitation of the 345 nm LMCT band of arthropod and molluskan Hcs. Experimental Section Sample Preparation. Hemolymph was extracted from B. canaliculatum (Marine Biological Laboratory, Woods Hole, MA) by an incision made across the foot. The turbid solution was centrifuged at low speed to remove debris and dialyzed against 50 mM Na2CO3 buffer (pH ) 9.8) and stored at 4 °C. The hemolymph of L. polyphemus (Marine Biological Laboratory, Woods Hole, MA) was extracted by syringe from the heart. The solution was filtered and centrifuged at low speed to remove gelatin-like clotting aggregates. The supernatant was dialyzed against 100 mM Tris-HCl buffer (pH 8.9) and stored at 4 °C. Absorption and Fluorescence Spectroscopy. A Hitachi U-2010 spectrophotometer was used to obtain steady-state absorption spectra. Emission spectra were recorded on a PerkinElmer LS 50-B Luminescence Spectrometer. Transient Absorption Spectroscopy. The experimental apparatus used in the femtosecond transient absorption studies has been described previously.29 The output pulses from a regeneratively amplified titanium:sapphire laser system (Spectra Physics, 1 kHz repetition rate) are used to pump an optical parametric amplifier (OPA, Spectra Physics), which can be tuned throughout the visible and UV spectral regions. The OPA output pulses used in this work were 200 fs in duration with a center wavelength of 340 or 375 nm. The OPA output was split into pump and probe beams and allowed to travel different path lengths before being recombined on the sample. The path length of the pump arm was varied using a computer controlled delay stage. Neutral density filters were used to attenuate the intensities

J. Phys. Chem. B, Vol. 105, No. 7, 2001 1479 of the pump (50 nJ) and probe (2 nJ) beams to avoid permanent photobleaching of the sample. The intensity of the probe beam, after traversing the sample, was measured using a photodiode and lock-in amplifier detection system. The Hc samples were concentrated in an Amicon ultrafiltration cell to achieve an optical density of 0.3-0.4 at 340 or 375 nm when measured in a 1 mm path length quartz cell. Photoacoustic Calorimetry. The experimental design and method of data analysis used here are similar to those described previously by Simon and co-workers,30 with the exception that femtosecond excitation pulses were used in this work. The concentrations of Hc and the reference compound, bromocresol purple (BCP), were adjusted to achieve an optical density of 0.1 at 340 nm for a 1 cm path length quartz cell. The samples were flowed through a 1 cm cell and were excited by 5 µJ, 200 fs pulses at 340 nm. An ethylene glycol bath chilled by a coldfinger was used to control the sample reservoir temperature. A thermocouple placed near the beam path inside the sample cell was used to measure the sample temperature. The excitation pulses were generated by the laser system described above. The photoacoustic signal was detected perpendicular to the excitation source by a 1 MHz piezoelectric transducer (Panametrics A103.S) attached to the sample cell with a thin layer of silicon grease. The signal was preamplified (Panametrics 5660b) and recorded by a digital oscilloscope (LeCroy 9450AM). A thorough review of the theory and applications of timeresolved photoacoustic calorimetry is given by Braslavsky and Heibel.31 The internal energy, and potentially the volume, of a molecule is increased when the molecule absorbs light. This energy can, in turn, be transferred to the solvent around the molecule. An acoustic wave is generated as the solvent expands from this energy transfer; the amplitude of the wave is related to the quantum yields for the nonradiative decay channels accessed by the excited molecule. The amplitude of the signal, S, is a function of ∆Vth, the change in volume of the solvent due to thermal expansion, and ∆Vm, the difference in volume between the products and reactants. The thermal contribution to S is given by

∆Vth ) (β/CpF)Q

(1)

where β is the thermal expansion coefficient of the solvent, Cp is the heat capacity of the solvent, F is the density of the solvent, and Q is the thermal energy released from the reactant. The photoacoustic signal can be expressed as

S ) κ(β/CpF)Q + ∆Vm

(2)

where the constant κ is unique to the instrument response function, which relates the amount of heat released to the cell geometry. For a reference compound (e.g., BCP) which is known to both convert all the absorbed energy into heat and undergo a negligible molecular volume change, ∆Vm ) 0. The ratio of S (the heat released by an arbitrary molecule) to Sref, φ, is then

φ ) S/Sref ) Q/Ehν + ∆Vm/(β/CpF)Ehν

(3)

where Ehν is the photon energy. Rearranging eq 3 gives

φEhν ) Q + ∆Vm/(β/CpF)

(4)

Q and ∆Vm are temperature-independent while the variables φ and β/CpF are temperature-dependent.32 A plot of φEhν versus (β/CpF)-1 will yield a slope and an intercept equal to ∆Vm and Q, respectively. Therefore, temperature-dependent photoacoustic

1480 J. Phys. Chem. B, Vol. 105, No. 7, 2001

Floyd et al.

Figure 3. Photoacoustic calorimetry signal of oxy hemocyanin (- -) and bromocresol purple (s) collected at 22.5 °C (top) and 10.1 °C (bottom) using 340 nm excitation.

TABLE 1: Temperature-Dependent Values of (β/CpG)-1 and OEhνa temperature (°C)

φEhν (kJ/mol)

(β/CpF)-1 (kJ/ml)

10.1 13.3 17.2 22.5

261 286 320 303

46.9 32.7 23.9 18

a

Figure 4. Dependence of φEhν on (β/CpF)-1 at various temperatures. The data are shown superimposed with a linear fit function (R2 ) 0.8) described by eq 4, with values for Q of 350 ( 15 kJ/mol and ∆Vm of -2 mL/mol.

Values for β, Cp, and F were taken from ref 32.

measurements allow for the determination of the molecular volume change and the nonradiative energy release that occurs on the time scale detected by the transducer. The transducer used in these experiments is capable of resolving kinetics that occur on a time scale of nanoseconds to a few hundred nanoseconds. Kinetics that occur on a faster time scale are not resolved but appear to occur instantaneous. To characterize the molecular volume changes and thermal energy release that occurs after photoexcitation of oxy Hc, we took photoacoustic measurements at 10.1, 13.3, 17.2, and 22.5 °C. Results Absorption and Emission Spectroscopy. The absorption spectrum of oxy Hc from B. canaliculatum is shown in Figure 2. An identical spectrum was recorded for oxy Hc from L. polyphemus (not shown). No fluorescence emission was observed following excitation of the 345 or 570 nm LMCT bands of oxy Hc at room temperature. Photoacoustic Calorimetry. Figure 3 shows characteristic photoacoustic signals for BCP and oxy Hc recorded at 10.1 and 22.5 °C following excitation at 340 nm. The isothermal expansion of water decreases significantly with decreasing temperature in the range tested. As a result, the amplitudes of the measured photoacoustic signals decrease as the temperature is lowered. At 22.5 °C, the BCP photoacoustic signal is larger than the oxy Hc signal, while at 10.1 oC, the oxy Hc signal is bigger then the BCP signal. The temperature-dependent ratio of Hc to the reference signal indicates that there is a molecular volume component to the photoacoustic signal that must be analyzed using eq 4. The photoaoustic data obtained at the four temperatures studied are given in Table 1. The data in Table 1 was used to construct a plot of φEhν versus (β/CpF)-1, as shown in Figure 4. The slope of a linear least-squares fit to the data gives a value for Q of 350 ( 15 kJ/mol and ∆Vm of -2 mL/ mol (∼3.5 Å3/active site). The 340 nm excitation used in this

Figure 5. One-color pump-probe transient recorded following excitation of oxy hemocyanin at 340 nm. The data are shown superimposed with a fit function (solid curve) that is described by a two-exponential model with time constants of 1.7 ( 0.2 and 80 ( 10 ps. The inset shows the decay dynamics plotted over the first 10 ps following excitation.

work corresponds to an energy of 352 kJ/mol. Thus, the photoacoustic measurements demonstrate that approximately 100% of the energy absorbed by the oxy Hc sample at 340 nm is released as heat, with an experimental error of roughly 4%. The lack of any temporal shift between the Hc and BCP photoacoustic waves indicates that the heat is released on the subnanosecond time scale.33 Femtosecond Transient Absorption Spectroscopy. Figure 5 shows the one-color absorption transient obtained with Hc from B. canaliculatum. The transient was obtained at room temperature with 200 fs pump and probe pulses centered at 340 nm. Identical dynamics were observed for L. polyphemus Hc (results not shown). In both cases, following the formation of an instantaneous bleach, the transients exhibit a recovery that is well described by a two-exponential model with time constants of 1.7 ( 0.2 and 80 ( 10 ps. The transients exhibit a residual bleach of roughly 5% of the total amplitude for probe delay times from 500 to 800 ps (the time limit of our experiment). The inset to Figure 5 shows the decay dynamics

Radiationless Decay in Photoexcited Hemocyanins

Figure 6. One-color pump-probe transient recorded following excitation of oxy hemocyanin at 340 nm (top) and 375 nm (bottom). Both transients are shown superimposed with a fit function that is described by a two-exponential model. The time constants used to model the 375 nm transient are 610 ( 100 fs and 80 ( 10 ps; the values for the 340 nm fit function are the same as those described for the data shown in Figure 5.

plotted over a shorter time range. The time scales and amplitudes of the decay components and the value of the offset were observed to be independent of excitation pulse energy over the range of 10-400 nJ. Figure 6 shows the absorption transient obtained with B. canaliculatum Hc using 375 nm pump and probe pulses plotted along with the 340 nm transient over a 100 ps time range. Two features stand out upon caparison of the data sets. The 375 nm transient exhibits a much faster initial amplitude decay than the 340 nm transient, and the transients converge at a probe delay time of roughly 70 ps. At longer delay times (not shown), the transients exhibit identical decay dynamics. A two-exponential model with time constants of 610 ( 100 fs and 80 ( 10 ps and a residual offset of 5% are used to describe the 375 nm data. Discussion Our initial studies of Hc were motivated, in part, by a lack of complete understanding of the fluorescence properties exhibited by deoxy and oxy Hc. Previous spectroscopic studies performed on Hc samples prepared in both states have shown that the fluorescence yield for oxy Hc, following excitation into the protein absorption band (which is centered at 280 nm), is quenched by a factor of ∼0.13-0.25 in comparison to that observed for deoxy Hc.34-36 The maximum of the fluorescence emission from the protein is centered at ∼330 nm and overlaps significantly with the 345 nm absorption band of oxy Hc. As a result, the fluorescence quenching has been attributed to a nonradiative electronic energy transfer reaction involving the initially excited protein residues (donors) and the oxygenated metal center (acceptor).34-36 An intriguing aspect of these studies is that after the proposed energy-transfer reaction, no significant room temperature fluorescence emission from the metal site was observed. Furthermore, as discussed in the results section of this work, no fluorescence emission from the 345 nm LMCT state of oxy Hc at room temperature was detected. These observations are a strong indication that the LMCT state relaxes primarily via a nonradiative mechanism, which would have to occur on a time scale (subnanosecond) that competes favorably with nanosecond (or longer) fluorescence dynamics. The photoacoustic and transient absorption experiments support this nonradiative model.

J. Phys. Chem. B, Vol. 105, No. 7, 2001 1481 As discussed above, the results of the photoacoustic experiments demonstrate that oxy Hc releases essentially all of the absorbed photon energy as heat on the subnanosecond time scale when the 345 nm LMCT band is excited. Furthermore, the nearly complete recovery of the bleach signal (5% offset) observed at a probe delay of 500 ps in the transient absorption experiments (see Figure 5) is an indication that at least 95% of the photoexcited molecules return to the ground state on the subnanosecond time scale. Taken together, these results provide conclusive evidence that the oxy Hc LMCT state does not undergo radiative decay to a large extent following photoexcitation. Photoexcitation of oxy Hc at 340 nm promotes an electron from a peroxide π orbital to a half-filled copper dx2-y2 orbital. In principle, the subsequent ground-state recovery could occur via radiative or nonradiative processes. Since the preceding analysis rules out radiative decay as the primary mechanism of decay, the decay components observed in Figures 5 and 6 can be assigned to nonradiative processes. However, the interpretation of the exact nature of the nonradiative processes becomes complicated due to the fact that both internal conversion and photoexcitation leading to photodissociation of O2 are valid pathways. Photodissociation and any subsequent geminate recombination of O2 with the metal center would be manifested as a bleach recovery in the absorption transient signal similar to the signal that would arise from internal conversion. Hence, the shape of the absorption transient signal measured is consistent with both possible pathways and does not help distinguish between the two pathways. The reported two-exponential decay detected in the femtosecond transient absorption experiment could simply reflect a distribution of geminate recombination rates. The validity of this purely photodissociative model can be examined by considering the two different wavelengths used to record absorption transients, 375 and 340 nm. The time scales for geminate recombination are not expected to be dependent on the excitation (photolysis) wavelength, as has been demonstrated from the previous work on heme proteins.26,37 For both oxy Hc proteins, the initial decay component is significantly faster at 375 versus 340 nm (fast 610 fs vs fast 1.7 ps), whereas the secondary decay component (slow 80 ps) and the residual offset (5%) remain unchanged with wavelength. The dependence of the value of the fast decay component on the excitation wavelength clearly demonstrates that it does not arise from geminate recombination of O2. Instead, it is assigned to a combination of excited-state vibrational relaxation and internal conversion processes. The resulting more complex model can be summarized as follows. The 340 nm excitation pulses populate the LMCT state with excess vibrational energy. This population undergoes rapid thermalization in the excited state before relaxing to the ground state via internal conversion; the 1.7 ps decay component is therefore a measure of both processes. As the excitation is tuned to the red portion of the 345 nm band, the LMCT state is populated with less excess vibrational energy. The 610 fs decay component measured at 375 nm is attributed primarily to the internal conversion process, which contains minimal contributions from excited-state vibrational relaxation at this wavelength. Again, at 340 nm the 1.7 ps decay component contains contributions from internal conversion (610 fs) and vibrational relaxation. On the basis of this argument, it is concluded that the LMCT state in oxy Hc decays to the ground state with a time constant of ∼610 fs following photoexcitation. This model is consistent with the extremely small fluorescence yield observed for oxy Hc: the

1482 J. Phys. Chem. B, Vol. 105, No. 7, 2001 subpicosecond internal conversion reaction competes favorably with radiative processes (which typically occur on the nanosecond time scale or longer) that contribute to the decay of the LMCT state. The interpretation of the slow phase of signal decay is less certain at this point. The excitation wavelength-independence of the time scale and amplitude of the 80 ps decay component suggests that it does not arise purely from electronic or vibrational relaxation processes. In light of this, two models are proposed which support the idea that the 80 ps component is associated with the kinetics arising from the recombination of O2 with the metal center following photo- and/or thermal dissociation. Before presenting these models, it is important to note that in both models the 610 fs decay component is still attributed to internal conversion and that photoexcitation of the oxy Hc 345 nm absorption band leads to the formation of the LMCT state with a quantum yield of 1. In the first model, the LMCT state is deactivated via two channels: internal conversion and photodissociation. Following excitation, a fraction of the photoexcited molecules undergoes direct relaxation to the ground state on the 610 fs time scale (internal conversion), whereas the remaining molecules return to the ground state following O2 dissociation and recombination with the metal center. The 80 ps component would then be assigned to dynamics associated with geminate recombination of O2 to the metal center, and the residual bleach (5%) would arise from the fraction of O2 molecules that do not recombine on the time scale of the experiment. Since there is no apparent delay in the onset of the 80 ps decay component, the transition from the LMCT state to the ligand-dissociative state must occur on the sub 100 fs time scale (time resolution of the experiment). The second model proposes that photodissociation does not occur but that the majority of photoexcited molecules undergoes nonradiative decay to the ground state on the 610 fs time scale. This rapid relaxation process would result in the formation of a “hot” ground state. Ligand loss would then occur via a thermal dissociative process from the hot ground state in competition with vibrational relaxation. The 80 ps decay component would reflect a combination of ground-state vibrational relaxation and O2 recombination dynamics. The major difficulty in assignment of the 80 ps decay component at this point is that Hc does not possess any spectroscopic signatures that correspond to the unligated species, as is the case for Hb and Mb, which we can monitor following photoexcitation. As a result, we are unable to determine definitively if any degree of ligand dissociation (thermal or photochemical) has been detected in the present study. The experiments conducted here only allow the dynamics associated with the reformation of the oxy Hc ground state to be monitored, which can occur via internal conversion, thermalization, and/ or ligand recombination processes after photoexcitation. Nevertheless, the possibility that we have observed O2 recombination in the present study leads us to consider what can be learned about the mechanism of ligand binding in Hc. As noted above, we are particularly interested in determining if our work can provide insight into the mechanism by which the active-site protein environment regulates ligand binding and release. Results from transient absorption studies indicate that breakage of the heme-ligand bond in Hb takes place in less than 50 fs with a quantum yield of 1 and that the ligand recombination mechanism varies depending on the nature of the ligand.24 For HbCO, CO geminate recombination is exponential and takes place on the 20 ns time scale.38 Conversely, geminate recombination of NO is nonexponential and can be described by a

Floyd et al. two-exponential model with time constants on the order of 10 and 60 ps.25 As pointed out by Martin and co-workers, nonexponential rebinding behavior can arise, in principle, from the presence of multiple protein conformations (substates) that relax with different rates or from a time-dependent energy barrier for geminate recombination that arises as the protein structure evolves to the deoxy conformation following ligand dissociation.25 The impact of either type of protein relaxation on the mechanism of ligand rebinding depends on the rate at which they occur relative to the rate of ligand binding. For Hb, significant rearrangement of the heme and its local environment occur over a range of time scales from 100 fs to 100 ps time scale following ligand photodissociation.25 Martin and coworkers suggest that these motions introduce a time-dependent barrier to ligand rebinding and are thus largely responsible for the NO nonexponential binding behavior.25 For Hc, subnanosecond motion of the active-site protein structure would be expected to induce nonexponential rebinding behavior in a manner similar to Hb. However, the results of the present study indicate that no significant protein relaxation processes occur on the subnanosecond time scale following dissociation of O2 in Hc. This conclusion is based on two observations. First, the signal decay component (80 ps time constant) that we attribute to O2 geminate recombination is exponential. Second, the absorption transient signal decay profile is identical for the arthropod and molluskan Hcs studied. If protein fluctuations were occurring on the picosecond time scale, we would expect to observe some deviation in the recombination kinetics exhibited by the arthropod and molluskan samples due to their significant differences in protein structure. Conclusions In the present study, photoacoustic calorimetry and femtosecond transient absorption spectroscopy have been used to characterize the dynamics that occur following photoexcitation of the 345 nm LMCT band of oxy Hc isolated from B. canaliculatum (mollusk) and L. polyphemus (arthropod). The results of the temperature-dependent photoacoustic studies reveal that following photoexcitation, both proteins release essentially all of the absorbed energy as heat on the subnanosecond time scale and undergo a small volume change of ∼3.5 Å3/active site. The transient absorption studies show that the LMCT state decays to the ground state on the 610 fs time scale and also suggest that photoexcitation leads to either photo- and/or thermal dissociation of O2, which ultimately recombines with the metal center on the 80 ps time scale. Additional studies are needed, however, to completely resolve this latter issue. Although the two proteins examined have different quaternary structures, the time-resolved data reveal that the active-site photophysics are identical for the two systems. This is consistent with the similarity of the active-site environment in various Hcs and suggests that interactions between the copper center and O2 are not strongly influenced by fluctuations of the protein environment in the region of the active site. Future studies comparing the dynamics exhibited by Hc model complexes to the data reported in this work may help to shed light on the role played by the active-site protein environment in mediating reversible ligand binding. Acknowledgment. This work was supported by the National Institutes of Health, MBRS funding program, GM08111 (M.D.E.), Duke University, and the North Carolina Biotechnology Center (J.D.S.). We thank Dr. Celia Bonaventura of the Duke Univer-

Radiationless Decay in Photoexcited Hemocyanins sity Marine Laboratories for providing a sample of hemocyanin from Limulus polyphemus. References and Notes (1) Lamy, J.; Lamy, J.; Weill, J. Arch. Biochem. Biophys. 1979, 193, 140. (2) van Holde, K. E.; Miller, K. I. Qu. ReV. Biophys. 1982, 15, 1. (3) Miller, K. I.; Schabtach, E.; van Holde, K. E. Proc. Natl. Acad. Sci. 1990, 87, 1496. (4) Cuff, M.; Miller, K. I.; van Holde, K. E.; Hendrickson, W. A. J. Mol. Biol. 1998, 278, 855. (5) Magnus, K. A.; Hazes, B.; Ton-That, H.; Bonaventura, C.; Bonaventura, J.; Hol, W. G. J. Proteins 1994, 19, 302. (6) Volbeda, A.; Hol, W. G. J. J. Mol. Biol. 1989, 209, 249. (7) Hazes, B.; Magnus, K. A.; Bonaventura, C.; Bonaventura, J.; Dauter, Z.; Kalk, K. H.; Hol, W. G. J. Protein Sci. 1993, 2, 597. (8) Larrabee, J. A.; Spiro, T. G. J. Am. Chem. Soc. 1980, 102, 4217. (9) Solomon, E. I.; Tuczek, F.; Root, D. E.; Brown, C. A. Chem. ReV. 1994, 94, 827. (10) Westermoreland, T. D.; Wilcox, D. E.; Baldwin, M. J.; Mims, W. B.; Solomon, E. I. J. Am. Chem. Soc. 1989, 111, 6106. (11) Ross, P. K.; Solomon, E. I. J. Am. Chem. Soc. 1991, 113, 3246. (12) Himmelwright, R. S.; Eickman, N. C.; LuBien, C. D.; Solomon, E. I. J. Am. Chem. Soc. 1980, 102, 5378. (13) Pate, J. E.; Ross, P. K.; Thamann, T. J.; Reed, C. A.; Karlin, K. D.; Sorrel, T. N.; Solomon, E. I. J. Am. Chem. Soc. 1989, 111, 5198. (14) Kitajima, N.; Fujisawa, K.; Fujimoto, C.; Moro-oka, Y.; Hashimoto, S.; Kitagawa, T.; Toriumi, K.; Tasumi, K.; Nakamura, A. J. Am. Chem. Soc. 1992, 114. (15) Magnus, K.; Ton-That, H.; Carpenter, J. In Bioinorganic Chemistry of Copper; Karlin, K., Tieklar, Z., Eds.; Chapman and Hall: New York, 1993; pp 143-150. (16) Solomon, E. I.; Lowery, M. D. Science 1993, 259, 1575. (17) Karlin, K.; Lee, D.; Humphreys, K. Pure Appl. Chem. 1998, 70, 855.

J. Phys. Chem. B, Vol. 105, No. 7, 2001 1483 (18) Comba, P.; Hilfenhaus, P.; Karlin, K. D. Inorg. Chem. 1997, 36, 2309. (19) Liao, Z.; Wang, J.; Shi, J.; Fan, B.; Xiao, L. J. Chim. Phys. 1995, 92, 668. (20) Lynch, W.; Kurtz, D.; Wang, S.; Scott, R. J. Am. Chem. Soc. 1994, 116, 11030. (21) Hong-Chang, L.; Dahan, M.; Karlin, K. Curr. Opin. Chem. Biol. 1999, 3, 168. (22) Rosca, F.; Kumar, A.; Ye, X.; Sjodin, T.; Demidov, A.; Champion, P. J. Phys. Chem. A 2000, 104, 4280. (23) Petrich, J. W.; Martin, J. L.; Houde, D.; Poyart, C.; Orszag, A. Biochemistry 1987, 26, 7914. (24) Petrich, J. W.; Poyart, C.; Martin, J. L. Biochemistry 1988, 27, 4049. (25) Petrich, J.; Lambry, J. C.; Kuczera, K.; Karplus, M.; Poyart, C.; Martin, J. L. Biochemistry 1991, 30, 3975. (26) Walda, K.; Liu, X.; Sharma, V.; Magde, D. Biochemistry 1994, 33, 2198. (27) Chen, E.; Kliger, D. S. Inorg. Chim. Acta 1996, 242, 149. (28) Jackson, T.; Lim, M.; Anfinrud, P. A. Chem. Phys. 1994, 180, 131. (29) Pullen, S. H.; Edington, M. D.; Studer-Martinez, S. L.; Staab, H. A.; Simon, J. D. J. Phys. Chem. A 1999, 103, 2740. (30) Hanson, K. M.; Li, B.; Simon, J. D. J. Am. Chem. Soc. 1997, 119, 2715. (31) Braslavsky, S. E.; Heibel, G. E. Chem. ReV. 1992, 92, 1381. (32) Handbook of Chemistry and Physics; CRC Press: Boca Raton, FL, 1988-89; pp F4-F5. (33) Peters, K. S. Angew. Chem., Int. Ed. Engl. 1994, 33, 294. (34) Hristova, J.; Dolashka, P.; Stoeva, S.; Voelter, W.; Salvato, B.; Genov, N. Spectrochim. Acta 1997, 53, 471. (35) Beltramini, M.; di Muro, P.; Rocco, G. P.; Salvato, B. Arch. Biochem. Biophys. 1994, 313, 318. (36) Shaklai, N.; Daniel, E. Biochemistry 1970, 9, 564. (37) Petrich, J. W.; Martin, J. L. Chem. Phys. 1989, 131, 31. (38) Jones, C. M.; Ansari, A.; Henry, E. R.; Christoph, G. W.; Hofrichter, J.; Eaton, W. A. Biochemistry 1992, 31, 6692.