©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Microsecond Generation of Oxygen-bound Cytochrome c Oxidase by Rapid Solution Mixing (*)

Satoshi Takahashi , Yuan-chin Ching , Jianling Wang , Denis L. Rousseau (§)

From the (1) From AT& Bell Laboratories, Murray Hill, New Jersey 07974

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Current understanding of the oxygen reduction and proton translocation processes in cytochrome c oxidase is largely derived from the data obtained by a nonphysiological method for initiating the catalytic reaction: photolyzing carbon monoxide (CO) from the CO-inhibited enzyme in the presence of oxygen (O). However, considerable evidence suggests that the use of CO introduces artifacts into the reaction mechanism. We have therefore developed a rapid solution mixer with a mixing time of 20 µs to study the catalytic reaction by directly mixing the enzyme with Owithout using CO. Unexpectedly, the resonance Raman scattering detected for the first 120 µs after the mixing show that the CO influences neither the structure of the primary oxy-intermediate, its rate of decay, nor the rate of oxidation of cytochrome a. This implies that CO has an effect on the later stages of the catalytic process, which may involve the proton translocation steps, and calls for the re-examination of the catalytic process by using the direct mixing method. In addition, these results demonstrate the feasibility of using the rapid mixing device for the study of biological reactions in the microsecond time domain.


INTRODUCTION

Cytochrome c oxidase, the terminal enzyme in the mitochondrial electron transport chain, reduces Oto water in a binuclear center formed by cytochrome aand a nearby copper atom, Cu. Two additional redox centers, Cuand cytochrome a, supply electrons to the binuclear center. The enzyme harnesses the chemical energy released by the Oreduction to translocate four protons across the inner mitochondrial membrane and thus contributes to mitochondrial ATP synthesis (1, 2) . The investigation of the catalytic process of the enzyme by directly mixing the enzyme with O( direct mixing method) has been impossible, because with a conventional stopped flow apparatus, the mixing dead time (several milliseconds) is comparable with the enzyme's turnover time (3) . Dynamic information such as the structure and kinetics of the intermediates and the proton uptake and release kinetics, on which the present understanding of the catalytic mechanism is largely based (1, 2, 4, 5) has therefore been obtained by the CO-flash method: initiating the reaction by photolyzing carbon monoxide from the CO-inhibited enzyme in the presence of O(6) . This method was first developed by Gibson and Greenwood for transient absorption studies of intermediates generated at room temperature (6) and by Chance et al. for both absorption and EPR studies of intermediates trapped at cryogenic temperatures (7) . The CO-flash method has been utilized for several decades under the general assumption that the use of CO does not affect the catalytic process (4, 6, 7, 8, 9, 10, 11, 12, 13) .

The most recent applications of the CO-flash method were the resonance Raman studies of intermediates in the reaction of the enzyme with O(1, 5, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) . Identification of iron-oxygen stretching Raman lines from many of the key intermediates have enabled the elucidation of their active site structures and the determination of their formation and decay kinetics. The rate of electron transfer from cytochrome a to cytochrome awas determined by following the oxidation states of cytochrome a (19, 22) . These studies, along with site-directed mutagenesis studies of bacterial oxidases (25) , are helping to clarify the microscopic mechanism for oxygen reduction and proton translocation in the enzyme. It is important in such studies of the catalytic process to approach physiological conditions as closely as possible. However, initiation of the catalytic process by the CO-flash method is clearly nonphysiological, and, in fact, the question of the introduction of artifacts in the enzymatic reaction due to the photolytic initiation has been raised (5, 26) .

Both optical absorption and resonance Raman studies show that possible artifacts are induced by the CO-flash method, since the spectra of the enzyme at the end of the reaction depend on the method of initiation: the CO-flash method results in the ``resting'' enzyme and the direct mixing method results in the ``pulsed'' form (5, 26) . Recent experiments suggest that Cuis the first binding site for O(10, 11, 27, 28) , but it is known that the photolyzed CO coordinates to Cuwhere it is not released until a few microseconds (28) , a time scale similar to that for Obinding to cytochrome a( = 8 µs) (11) . Moreover, time-resolved resonance Raman measurements have shown that cytochrome ais unrelaxed until several microseconds after the photolysis of CO (29) . The CO-flash method could have a faster Oon-rate than that of the direct mixing method if the Ois already in the vicinity of active site prior to the CO dissociation. On the other hand, if the active site cannot accommodate two ligands, the presence of CO at Cucould delay Oassociation. Last, the structure of the reduced enzyme in the region of the binuclear center could be quite different if generated from a CO-bound form versus a ligand-free form (26) . These arguments suggest that anomalous results might be introduced by the use of CO especially in the early stages of the reaction. We have therefore developed a rapid solution mixer with a mixing/dead time of 20 µs and followed the first 120 µs of the reaction of cytochrome c oxidase with Oby resonance Raman scattering.


EXPERIMENTAL PROCEDURES

The time limit of a conventional stopped flow mixer (several ms) results from the need for relatively large solution volumes (10 µl) required for optical absorption. When the reaction is being probed by a laser beam to obtain resonance Raman or fluorescence spectra, large volumes are not necessary. Volume elements on the order of 10 µm 10 µm 10 µm (1 pl) are sufficient to obtain high quality spectra. Moreover, by using a small volume mixer with the ``continuous flow'' method, the flow speed can be made very high, which accelerates the generation of turbulence in the sample flow and thus promotes efficient mixing. Using these concepts, a method for mixing two solutions on the microsecond time scale to study chemical dynamics by fluorescence was first developed by Regenfuss et al. (30, 31) and subsequently used by Paeng et al. (32, 33) with resonance Raman scattering. We have adapted their general ideas and constructed a rapid mixing device with several modifications.

The mixing point in our rapid mixer is a simple pinhole nozzle as illustrated in Fig. 1( inset). Two solutions are introduced into the mixer co-axially but do not mix until they reach the pinhole, where the flow becomes turbulent due to the sudden increase of the flow speed and the geometry of the aperture. Since the mixing time depends on the particular nozzle, we picked a nozzle with a 20-µs mixing time for the time resolved measurements. In Fig. 1(plot) the mixing time of the nozzle was determined by comparing the intensities of Raman lines from HPO42- and HPO4- in the acid/base reaction between NaHPOand HCl. From this we determined that mixing was 90% complete in less than 20 µs. The nozzle aperture is 25 µm in diameter and 600 µm long. The holes in the stainless steel nozzles were made by laser drilling. The parent solutions were filtered to remove dust and transferred to gas-tight syringes with equal volumes (25 ml) where they were driven by a syringe pump at a rate of 0.64 ml/min per syringe. The time after mixing was estimated from the flow speed of the jet (43 µm/µs) which is calculated by assuming a homogeneous 25-µm diameter jet over the short distance (5 mm) between the entrance of the aperture of the nozzle and the longest time point reported here (120 µs). Time-resolved Raman scattering measurements are obtained by moving the focus point of the laser away from the nozzle exit along the sample jet. Full details of the mixing device will be described elsewhere.


Figure 1: The relative intensity of a Raman line from HPO42- is plotted as a function of time after mixing with HCl. Mixing is 90% complete in 20 µs. A schematic illustration of the rapid mixing device used for these measurements is shown in the inset and described in the text.



Cytochrome c oxidase was purified from beef heart by the method described by Yoshikawa et al. (34). The enzyme (95 µ M), dissolved in 50 m M sodium phosphate buffer (pH 7.4), was reduced by adding cytochrome c and sodium ascorbate to final concentrations of 5 µ M and 20 m M, respectively. The oxygen-saturated buffers were prepared by shaking the buffer solution vigorously under 1 atmosphere of either Oor Ogas. All the experiments were performed at room temperature.

Resonance Raman measurements were obtained by focusing the output from a Krypton ion laser on the sample jet. The Raman scattering was dispersed and measured by a single spectrometer equipped with a liquid Ncooled charge-coupled device camera. A holographic filter was used to remove the laser scattering.


RESULTS AND DISCUSSION

The Fe-Ostretching frequency of the O-bound enzyme generated by the CO-flash method was identified at 570 cmand had a lifetime of about 50 µs (14, 18, 21) . To locate the mode by our direct mixing method, we compared the resonance Raman spectra from the heme groups after mixing the fully reduced enzyme with Oor Osaturated buffer. As illustrated in Fig. 2, we detect a strong line at 570 cmfor Owhich shifts to 546 cmfor O, thus identifying this line as the Fe-Ostretching mode in exact agreement with the frequencies obtained by the CO-flash protocol. We conclude that any possible residual CO or unrelaxed protein in the CO-flash experiments has no effect on the structure of the Fe-Ounit of cytochrome a. We observed that the intensity of the Fe-Ostretching mode decayed to the background level within 100 µs (data not shown), also consistent with the CO-flash measurements (18, 21) .

An unexpected result of the CO-flash experiments was that cytochrome a was oxidized simultaneously with the decay of the primary Fe-Ointermediate (19, 22) . This result suggests that electron transfer from cytochrome a to oxy-cytochrome a( k 3 10s), where the iron atoms of the two hemes have a separation of 18 Å, occurs faster than electron transfer from Cuto oxy-cytochrome a( k 4 10s) where the oxygen points toward the Cuand the iron-copper distance is only 3-5 Å (35, 36). The electron transfer from cytochrome a to the binuclear center is known to be quite sensitive to the enzyme preparation (37) , and thus it, as well as the electron transfer dynamics between Cuand the cytochrome aheme, could be modulated by a conformational state set by the photodissociation process or the presence of photodissociated CO near the active site.

The electron transfer rate for the direct mixing method was estimated by following the oxidation state of cytochrome a (19, 22) in the high frequency resonance Raman spectra (Fig. 3). In the top trace (10 µs) the two lines in the electron density marker line () region (1356 and 1372 cm) show that the enzyme is largely in the reduced ligand-free state with the contribution from 1372 cmcoming from some O-bound cytochrome a. At 30 µs, the intense contribution at 1372 cmindicates that cytochrome ais almost completely O-bound. That the high intensity of the 1372 cmline at this time comes primarily from Ocoordination to cytochrome aand not oxidation of cytochrome a is confirmed by examining the 1600-1650 cmregion. The slight decrease in intensity at 1609 and 1624 cm, marker lines for reduced cytochrome a, with the concomitant increase in intensity at 1641 cm, a marker line for oxidized cytochrome a, indicates that only a small amount of electron transfer from cytochrome a to the binuclear site has occurred at this early time. At the next time point (50 µs) nearly half of cytochrome a has become oxidized, and at 80 µs some further oxidation of cytochrome a has taken place. No additional change is detected at 120 µs. Since cytochrome a and Cuare in equilibrium (38) , the fractional oxidation of cytochrome a is consistent with one electron transferred to the binuclear site. These results show two important features. First, Ocoordination to cytochrome aoccurs within the mixing time of our apparatus. Thus, the rapid coordination of Odoes not depend on it being in or near the heme pocket, as it might be in the CO-flash experiments. Second, the oxidation of cytochrome a occurs just as rapidly in the direct mixing experiments as it does in the CO-flash experiments and occurs simultaneously with the decay of the primary Fe-Ointermediate.


Figure 3: Time resolved resonance Raman spectra in the high frequency region obtained at several different delay times (as indicated) from the samples after directly mixing fully reduced cytochrome c oxidase with O saturated buffer. Experimental conditions are the same as those in Fig. 2, except for the use of a nozzle with a 20-µs mixing time, and the laser power was 80 milliwatts. Each spectrum was accumulated for 20 s.



The experiments reported here demonstrate the structural identity of the O-bound cytochrome c oxidase prepared by the direct mixing and the CO-flash methods. The kinetic properties, including the decay of the primary intermediate and the rate of oxidation of cytochrome a, are also the same as those found in the CO-flash experiments. Thus, the photodissociation of the CO does not affect these early events, although we cannot exclude the possibility that the Oon-rate is faster in the direct mixing method. Considering the reported differences at the end of the reaction between the direct mixing and the CO-flash methods (5, 26), there must be structural changes in the enzyme that will only become evident in the properties of the later intermediates which must be clarified by further investigation. It is especially important to delineate the properties of the later intermediates, since it has been shown that they are involved in the coupling between the redox processes and proton translocation (39). The experiments also demonstrate the feasibility of the rapid mixing device to address a problem of current biophysical interest. We expect that this method for the study of microsecond reaction dynamics will become important in several other fields of chemical physics and biophysics.


FOOTNOTES

*
This work was supported in part by Grant GM-48714 from the National Institute of General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 908-582-2609; Fax: 908-582-2451; E-mail: dlr@physics.att.com.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.