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200 µs Observed by a New Freeze-Quench Device
* Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan; and Division of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka Osaka 560-8531, Japan
Correspondence: Address reprint requests to Dr. Satoshi Takahashi or Prof. Isao Morishima, Dept. of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan. Tel.: 81-75-753-5921; Fax: 81-75-751-7611; E-mail: st{at}mds.moleng.kyoto-u.ac.jp or morisima{at}mds.moleng.kyoto-u.ac.jp.
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ABSTRACT |
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200 µs of the mixing-to-freezing time interval and observed the reaction between HRP and hydrogen peroxide (H2O2). The developed device consists of a submillisecond solution mixer and rotating copper or silver plates cooled at 77 K; it freezes the small droplets of mixed solution on the surface of the rotating plates. The ultraviolet-visible spectra of the sample quenched at 1 ms after the mixing of HRP and H2O2 suggest the formation of compound I. The electron paramagnetic resonance spectra of the same reaction quenched at 200 µs show a convex peak at g = 2.00, which is identified as compound I due to its microwave power and temperature dependencies. The absence of ferric signals in the electron paramagnetic resonance spectra of the quenched sample indicates that compound I is formed within 200 µs after mixing HRP and H2O2. We conclude that the activation of H2O2 in HRP at ambient temperature completes within 200 µs. The developed device can be generally applied to investigate the electronic structures of short-lived intermediates of metalloenzymes. |
INTRODUCTION |
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O) and a porphyrin 
cation radical. Compound I oxidizes a substrate molecule to become the next intermediate, compound II, which returns to the resting state by oxidizing another substrate molecule (Dunford, 1991
).
One of the unique characteristics of peroxidases is the rapid formation of compound I (107 M-1 s-1; Dunford, 1991). Unlike peroxidases, oxygen-binding heme proteins such as myoglobin react with H2O2 slowly (102 M-1 s-1) and do not form stable compound I (Matsui et al., 1999; Egawa et al., 2000). Poulos and Kraut (1980) proposed that the formation of compound I in peroxidases is promoted by the conserved His and Arg residues in the distal cavity of the heme. They assumed that the heme-peroxide complex called compound 0 was the precursor of compound I, whose O
O bond was cleaved with the aid of the distal His and Arg residues. Numerous mutagenesis studies verified the importance of these residues for compound I formation and supported the Poulus-Kraut model (Erman et al., 1993; Vitello et al., 1993; Newmyer and Ortiz de Montellano, 1995; Savenkova et al., 1998; Rodriguez-Lopez et al., 1996a,b, 2001).
Despite the accumulated evidence for the Poulus-Kraut model, the presence of compound 0 in peroxidases is suggested only by a few kinetic data including the low-temperature studies on horseradish peroxidase (HRP; Balny et al., 1987; Baek and Van Wart, 1989, 1992; Ozaki et al., 1998) and the study on the mutant of HRP (Rodriguez-Lopez et al., 1996b, 2001). The reaction between HRP and H2O2 at a cryogenic temperature indicates the saturation kinetics against the H2O2 concentration, which suggest the presence of an intermediate state before the formation of compound I (Balny et al., 1987; Baek and Van Wart, 1989, 1992). Baek and Van Wart further reported the low-temperature electronic absorption spectrum for the precursor, compound 0, which was later interpreted as the low-spin complex of heme with deprotonated peroxide anion (Baek and Van Wart, 1989, 1992; Harris and Loew, 1996). In contrast, the compound 0 species observed in the Arg-38
Leu (R38L) mutant of HRP is suggested as the high-spin complex of heme with neutral peroxide (Rodriguez-Lopez et al., 1996b, 2001). The low-temperature study that used polyethylene-glycolated HRP was rather consistent with the results of the R38L-mutant study (Ozaki et al., 1998). Thus, the properties of compound 0 that are suggested by these studies are not consistent.
A potentially powerful technique to characterize the kinetic and electronic properties of compound 0 is electron paramagnetic resonance (EPR) spectroscopy coupled with the rapid-quenching method. This method has been utilized to characterize the intermediate states of various metalloenzymes since Bray (1961) reported the first application. The limitation of this technique, however, is the relatively long dead time. The time interval between mixing and freezing is 5 ms, which is much longer than the estimated lifetime of compound 0 at room temperature. For example, the low-temperature results of Baek and van Wart (1989, 1992) can be extrapolated to room temperature to give a lifetime of 1 ms. Accordingly, an improvement in time resolution is necessary for the rapid-quenching technique to be used for the characterization of compound 0.
To observe the reaction dynamics of HRP using EPR spectroscopy, we developed a new freeze-quench device that can interrupt chemical reactions within 200 µs after mixing two solutions. Here we describe the design and performance of our device. The formation process of compound I in HRP was investigated using the device coupled with ultraviolet (UV)-visible and EPR spectroscopies. Our results demonstrate that the conversion of compound 0 to compound I is completed within 200 µs at room temperature.
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MATERIALS AND METHODS |
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). The activation of HRP by calcium and heme and the subsequent purification were conducted according to reported methods (Smith et al., 1990; Gazaryan et al., 1994) with some modifications (Nagano et al., 1996). The final RZ value was 3.2. The concentration of HRP was determined by the Soret extinction coefficient of 103 mM-1 cm-1 (Paul et al., 1953). All solutions for spectroscopic measurements contained 50 mM 3-morpholinopropanesulfonic acid (MOPS) at pH 7.0 unless otherwise stated.
Construction of the rapid mixing and freezing device
All parts of the mixing and freezing device were homemade. A block diagram of the constructed system is depicted in Fig. 1. For the quenching experiments at 200 µs (see Figs. 2, 3, and 6), the syringe pump was equipped with two 5-ml Hamilton syringes and supplied two solutions continuously at a flow rate of 4.2 ml min-1 per syringe. For the quenching experiments at 1 ms (see Figs. 4 and 5), 5- and 1-ml syringes were utilized at flow rates of 2 and 0.4 ml min-1, respectively. To reduce the sample waste, the sample solution was initially introduced into the sample loop (500 µl) and pushed by a buffer solution in the syringe (Fig. 1 a, syringe S2). The detailed design of the rapid mixer is indicated in Fig. 1 b. Two solutions were continuously introduced into the stainless mixing plate, which has a T-shaped mixing channel (Fig. 1 b, circle) that was drilled by Machida-Toolex (Tokyo, Japan). The plate was sandwiched by Teflon plates and tightened by metal blocks. The mixed solution flows through the exit channel (60-µm diameter; 200-µm length) and forms a jet.
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1000 rpm. The rapid mixer was held at a distance of 1 cm from the surface of the rotating disks, which were sprayed with the mixed solution. The frozen samples were transferred to liquid nitrogen during the rotation of the metal disks. After the freezing procedure, the powder samples were decanted and transferred manually to quartz cuvettes (1-mm inner thickness) or Suprasil tubes (4-mm inner diameter) immersed in liquid nitrogen for the optical absorption and EPR measurements, respectively. The sample powders were packed in the cuvettes or tubes, and the remaining liquid nitrogen was removed carefully. The procedures for the sample transfer were relatively easy due to the low viscosity and the specific gravity of liquid nitrogen. In contrast, the collection of sample powders from the isopentane bath was difficult and required a special technique (Tsai et al., 1998). To remove liquid oxygen from the EPR samples, the EPR tube containing the sample powders was moved to an isopentane bath cooled at 150 K, evacuated using a vacuum line, and sealed. Great care should be practiced to prevent the potential hazard of accumulating liquid oxygen in the liquid nitrogen bath. We used a minimum amount of cooled isopentane, which was prepared and handled inside a draft chamber.
We initially utilized the copper disks for the freezing of sample droplets; however, we found that the copper disks occasionally gave cupric signals in the EPR spectra. We therefore used silver disks for the preparation of EPR samples. We confirmed that the copper and silver disks gave almost comparable time intervals between mixing and freezing. The samples quenched at 200 µs (see Figs. 2, 3, and 6) and at 1 ms (see Figs. 4 and 5) were prepared using the silver and copper disks, respectively.
Optical absorption spectroscopy
Optical absorption spectra were recorded on PerkinElmer spectrometer (Lambda 19). The spectrometer was modified to incorporate an Oxford cryostat (DN1704) at the sample compartment. To observe the spectra of powder samples, we inserted the frosted quartz plates in the sample and reference beams. Furthermore, the intensity of the reference beam was reduced using a black block to compensate for the intensity reduction of the sample beam. The sample temperature was monitored by a thermocouple inserted directly in the cell. The slit width and the scan rate of the spectrometer were adjusted at 2 nm and 120 nm min-1, respectively. Although the inner thickness of the sample cells was 1 mm, the length should not be considered as the optical path length due to the powdery nature of the samples. The obtained spectra therefore reflect qualitatively the optical properties of the samples.
EPR spectroscopy
EPR spectra were measured on a Varian E-12 spectrometer equipped with an Oxford ESR-900 liquid helium cryostat. The microwave frequency was X-band (9.22 GHz), and the measurements were carried out with a modulation of 0.5 mT at 5 and 15 K. The microwave power was 5 mW.
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RESULTS AND DISCUSSION |
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and later redesigned by Takahashi et al. (1995, 1997). By using the mixer, we can achieve the mixing of two solutions within 100 µs. Furthermore, due to the high mixing efficiency of the mixer, we can reduce the speed of sample supply (4.2 ml min-1 for each solution), which makes practical operation of the new device much easier than the conventional ram system (>30 ml min-1 for each solution).
The second novel point of the developed device is the utilization of the "cold-block freezing" method for freezing the mixed solution. Since the first report by Bray (1961), the rapid-quenching method for the spectroscopic application has been limited to the "spray-freezing" method, that is, spraying droplets of mixed solution into liquid cryogens such as cooled isopentane. However, it has been impossible to reduce the time interval between the mixing and freezing to <5 ms using this method (Moodie et al., 1990; Saigo et al., 1993). The rapid freezing of biological specimens has been studied extensively in the field of electron microscopy (Robards and Steytr, 1985). It is known that the fastest freezing time can be achieved by impacting a specimen onto the polished surface of cold metal blocks (cold-block freezing). For example, Bald (1985) estimated that the cooling rate of biological specimens placed on a cooled copper plate at 78 K was >106 K s-1 at 10 µm inside of the sample from the contact surface. Thus, small water droplets with a diameter of several tens of micrometers can be cooled >100 K within 1 ms after contact with cooled copper plates.
Freezing dead time of the freeze-quench device
The freezing dead time of the device was first estimated using a reference reaction between horse-heart metmyoglobin and azide. Two solutions that contained metmyoglobin and azide were injected into the freeze-quench device to final concentrations of 100 µM and 800 mM, respectively, at a rate of 4.2 ml min-1 for each solution. The UV-visible spectrum of the quenched sample was measured at 120 K (Fig. 2, trace A). A broad Soret band was observed at 413 nm, where the two Soret bands of metmyoglobin (410 nm for the frozen powers; Fig. 2, trace E) and azide-bound metmyoglobin (421 nm for the frozen powders; Fig. 2, trace D) were overlapped. The datum indicates that the device trapped the azide-binding reaction before its completion. Upon the increase in the sample temperature from 120 to 240 K, the Soret maximum gradually moved to 421 nm as shown in Fig. 2, traces B–D; this indicates the conversion to the azide-bound form. The changes in the absorption peaks in the visible region are also consistent with the conversion of metmyoglobin (500 nm) to the azide-bound form (541 and 577 nm). The disappearance of metmyoglobin demonstrates the perfect mixing of the two solutions before the freezing. We can roughly estimate that the amount of the azide-bound form in the spectrum taken at 120 K (Fig. 2, trace A) was 50% via calculation of the spectral overlap of the azide-bound and free (Fig. 2, traces D and E, respectively) forms of metmyoglobin. Because the half-value period of metmyoglobin in the presence of 800 mM azide is 120 µs at 2123°C (Antonini and Burunori, 1971), the estimated dead time of the freeze-quench device is 120 µs. We also confirmed that 1 ms of the mixing-to-freezing time interval can be obtained by reducing the rates of sample supply to 0.4 and 2 ml min-1 for metmyoglobin and azide solutions, respectively (data not shown).
We further confirmed the mixing-to-freezing time interval using another fast reaction between myoglobin and oxygen. The reduced form of myoglobin (200 µM) in 50 mM MOPS buffer at pH 7.0 was prepared using dithionite (5 mM) and was anaerobically transferred to one of the syringes. The other syringe was filled with the same buffer saturated with air (280 µM oxygen). Both solutions were injected into the freeze-quench device at a rate of 4.2 ml min-1. Fig. 3, trace A indicates the optical absorption spectrum of the quenched sample at 130 K. Two Soret peaks at 437 and 420 nm indicate the presence of the ferrous deoxygenated (Fig. 3, trace E) and the ferrous oxygen-bound forms of myoglobin, respectively. The deoxy peak at 437 nm gradually disappeared upon the increase in the sample temperature to 200 K (Fig. 3, trace B), 210 K (trace C), and 225 K (trace D) and converted to the oxygen-bound form. These observations indicate that the device can mix two solutions completely and freeze the mixed solution before the completion of the oxygen binding to myoglobin. By calculating spectral overlap, we estimate the amount of the oxygen-bound form in the spectrum taken at 130 K was 40%, which corresponds to 200 µs of the time interval after the mixing of 140 µM oxygen at 20°C (Antonini and Burunori, 1971).
Although the determined time intervals between mixing and freezing should be considered as rough estimates, the two different reactions gave consistent time constants of 120200 µs. We therefore conclude that the mixing-to-freezing time interval of the current device is 200 µs. This is roughly an order shorter than those achieved by conventional techniques (5 ms).
UV-visible spectra of the reaction mixture between ferric HRP and H2O2
Ferric HRP and H2O2 were mixed to final concentrations of 20 and 400 µM, respectively, and frozen 1 ms after mixing using the developed freeze-quench system. We used 50 mM MOPS buffer adjusted at pH 7.0, because the buffer is known to be stable to the pH change at the low temperature (Orii and Morita, 1977). The UV-visible spectrum of the reaction mixture was measured at 114 K (Fig. 4, trace A) and showed a Soret absorption at 401 nm and a broad featureless band in the visible region. Notably, the spectrum at 114 K possessed a shoulder at 650 nm (Fig. 4, inset) that is characteristic of compound I (Dolphin et al., 1971; Dunford, 1991). The spectra suggest that the intermediate observed at 1 ms is compound I.
We next examined the temperature dependence of the optical absorption spectra. The UV-visible spectrum of compound I was unchanged up to 202 K (Fig. 4, traces B and C), began to change at around 230 K (traces D and E), and at 256 K converted to a new species that has a Soret band at 417 nm and Q-bands at 529 and 555 nm (trace F). The spectrum is similar to that of compound II (419, 528, and 553 nm) when generated at room temperature and frozen by the developed device (spectrum not shown) and suggests the reduction of compound I to compound II at the higher temperature. The sample further indicates the change of the Soret maximum to 406 nm upon the increase in temperature to 264 K (Fig. 4, trace G), which is similar to that of the resting enzyme.
We noticed that the choice of buffer systems is important for obtaining consistent results at room and cryogenic temperatures. The same quenching experiment of the HRP-H2O2 reaction dissolved in a 50 mM sodium phosphate buffer was conducted. As indicated in Fig. 5 (trace A), the quenched sample within 1 ms contained both compound I and compound II at 150 K as is evident from a peak at 650 nm and a shoulder at 419 nm. The latter feature grew as the temperature increased (Fig. 5, traces B and C). However, the visible peaks for compound II that were apparent at 230 K (Fig. 5, trace D) were observed at 539 and 571 nm, which are different from the peaks observed when the MOPS buffer was used (529 and 555 nm). A peak at 670 nm that appeared at 260 K (Fig. 5, trace E) suggests the modification of the heme macrocycle (Nakajima and Yamazaki, 1980) probably due to the high concentration (10 mM) of H2O2. We suggest that the fast formation and modified absorption spectrum of compound II that were observed in the phosphate buffer were caused by the pH change of the phosphate buffer at the cryogenic temperature. Orii and Morita (1977) reported that the pH of sodium phosphate buffer can be decreased by 4 units on freezing. Furthermore, an increase in the redox potential for the compound I/compound II couple at the low-pH condition was reported (Hayashi and Yamazaki, 1979). Thus, the low-temperature reaction of HRP in the phosphate buffer is modified from that at room temperature.
EPR spectra of the reaction mixture between ferric HRP and H2O2
To observe the faster phase of the reaction, we next quenched the sample at 200 µs after mixing HRP and H2O2 in the MOPS buffer. Because it is difficult to differentiate compound I from the resting state by optical absorption spectra for powder samples, we examined the quenched sample using EPR spectroscopy. As shown in Fig. 6 (trace B), the EPR spectrum of the quenched sample observed at 5 K was characterized by a convex peak at g = 2.00. The convex signal at g = 2.00 was identical to the signal observed for compound I that was prepared by manually freezing the mixture of HRP and H2O2 in the MOPS buffer (the interval between mixing and freezing was 30 s; Fig. 6, trace D). The convex peak disappeared upon the increase in temperature to 15 K (Fig. 6, trace C), which was in agreement with the relaxation broadening of the signal for compound I (Aasa et al., 1975; Schultz et al., 1979). Furthermore, the convex peak was not saturated up to 50 mW of microwave power (not shown), which was also in accordance with the signal of compound I (Aasa et al., 1975; Schultz et al., 1979). We conclude that the intermediate state observed in the quenched sample at 200 µs is compound I. We also confirmed that the sample quenched at 1 ms after being mixed in MOPS and phosphate buffers gave only the spectrum of compound I (not shown).
We could not observe signals from compound 0 in the quenched sample at 200 µs. Previous studies revealed that the iron-peroxide complex of sperm whale myoglobin mutants, in which the distal His was replaced with Val or Gln, showed ferric low-spin signals at g values of 2.29, 2.16, and 1.91 (Brittain et al., 1997). On the contrary, the ferric high-spin state was proposed as the electronic state of compound 0 in the R38L mutant of HRP (Rodriguez-Lopez et al., 1996b). However, the small signals observed in the high-spin region (g = 6) for the quenched sample (Fig. 6, trace B) arose from the remaining resting enzyme (trace A). The ferric low-spin hemes were also absent in the EPR spectrum observed at 15 K (Fig. 6, trace C), where the low-spin signals should be enhanced. The absence of even a trace amount of compound 0 at 200 µs indicates that the lifetime of compound 0 at ambient temperature is <100 µs.
The current observation resolves conflicting information regarding the lifetime of compound 0. The low-temperature results by Baek and van Wart (1989, 1992) can be extrapolated to room temperature to give 1 ms of the lifetime. In contrast, Balny et al. (1987) estimated 20 µs from the similar low-temperature experiments. The discrepancy was likely caused by the difficulty in eliminating the artifacts of low-temperature experiments such as the pH changes of buffers as observed in this and previous studies (Orii and Morita, 1977). The destabilization of proteins in the methanol-based cryosolvents was also reported (Virden et al., 1990). The rapid-quenching method is free from these artifacts, because the reaction is initiated and conducted at room temperature in a normal buffer system. It is interesting to note that the estimated lifetime of compound 0 (<100 µs) is comparable with the timescale of OO bond cleavage of dioxygen (O2) conducted by cytochrome oxidase (100 µs; Han et al., 2000). The structures surrounding the H2O2 or O2 binding sites of these proteins should play essential roles in the very fast activation of the OO bond. The distal structures were clarified in the atomic resolution for compounds I and II of HRP (Bergund et al., 2002). The kinetic and spectroscopic investigations on a submillisecond timescale using the developed device are important to comprehend the molecular mechanism of heme peroxidases and other metalloenzymes.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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S. Yoshioka's present address is Dept. of Biochemistry, Vanderbilt University, Nashville, TN 37232-0146.
Submitted on May 28, 2002; accepted for publication October 3, 2002.
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H. Kumita, K. Matsuura, T. Hino, S. Takahashi, H. Hori, Y. Fukumori, I. Morishima, and Y. Shiro NO Reduction by Nitric-oxide Reductase from Denitrifying Bacterium Pseudomonas aeruginosa: CHARACTERIZATION OF REACTION INTERMEDIATES THAT APPEAR IN THE SINGLE TURNOVER CYCLE J. Biol. Chem., December 31, 2004; 279(53): 55247 - 55254. [Abstract] [Full Text] [PDF]
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V. Schunemann, F. Lendzian, C. Jung, J. Contzen, A.-L. Barra, S. G. Sligar, and A. X. Trautwein Tyrosine Radical Formation in the Reaction of Wild Type and Mutant Cytochrome P450cam with Peroxy Acids: A MULTIFREQUENCY EPR STUDY OF INTERMEDIATES ON THE MILLISECOND TIME SCALE J. Biol. Chem., March 19, 2004; 279(12): 10919 - 10930. [Abstract] [Full Text] [PDF]
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T. Egawa, S. Yoshioka, S. Takahashi, H. Hori, S. Nagano, H. Shimada, K. Ishimori, I. Morishima, M. Suematsu, and Y. Ishimura Kinetic and Spectroscopic Characterization of a Hydroperoxy Compound in the Reaction of Native Myoglobin with Hydrogen Peroxide J. Biol. Chem., October 24, 2003; 278(43): 41597 - 41606. [Abstract] [Full Text] [PDF]
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