Time-resolved flow-flash FT-IR difference spectroscopy: the kinetics of CO photodissociation from myoglobin revisited
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Fourier-transform infrared (FT-IR) difference spectroscopy has been proven to be a significant tool in biospectroscopy. In particular, the step-scan technique monitors structural and electronic changes at time resolutions down to a few nanoseconds retaining the multiplex advantage of FT-IR. For the elucidation of the functional mechanisms of proteins, this technique is currently limited to repetitive systems undergoing a rapid photocycle. To overcome this obstacle, we developed a flow-flash experiment in a miniaturised flow channel which was integrated into a step-scan FT-IR spectroscopic setup. As a proof of principle, we studied the rebinding reaction of CO to myoglobin after photodissociation. The use of microfluidics reduced the sample consumption drastically such that a typical step-scan experiment takes only a few 10 ml of a millimolar sample solution, making this method particularly interesting for the investigation of biological samples that are only available in small quantities. Moreover, the flow cell provides the unique opportunity to assess the reaction mechanism of proteins that cycle slowly or react irreversibly. We infer that this novel approach will help in the elucidation of molecular reactions as complex as those of vectorial ion transfer in membrane proteins. The potential application to the oxygen splitting reaction of cytochrome c oxidase is discussed.
KeywordsCarbonmonoxymyoglobin Cytochrome c oxidase Microfluidics Step-scan spectroscopy Vibrational spectroscopy
Myoglobin is a monomeric oxygen transporting and storing protein of muscle tissue. The protein is well characterised and considered the hydrogen atom of biology . The structure of the protein comprises eight helices A–H. The cofactor, heme b, is embedded in a hydrophobic pocket between helices E and F and ligated to the protein via a proximal histidine residue. The central ferric iron of heme b is able to bind oxygen and other small molecules, like carbon monoxide or nitric oxide. Substrate binding and rebinding kinetics after photolysis of myoglobin have been intensively studied, e.g. by X-ray crystallography [2, 3], time-resolved UV/Vis spectroscopy [4, 5] and time-resolved infrared spectroscopy [6, 7]. Instead of the natural ligand oxygen, infrared (IR) spectroscopy mostly uses the polar CO as IR probe which is photo-labile with a quantum efficiency of photolysis of close to unity . Photodissociation of carbonmonoxymyoglobin (Mb-CO) yields a short-lived photoproduct, which relaxes to the deoxy form of myoglobin (deoxy-Mb) . Deoxy-Mb exhibits several structural differences to Mb-CO, in particular heme-doming due to the out-of-plane movement of the heme iron, protein backbone displacements of helices B, C and E and, to a lesser extent, of helices F and G. As a consequence, single amino acids change their positions: H64 (sperm whale numbering) moves into the heme pocket, and the side chain of L29 rotates [2, 9]. The photo-dissociated CO is able to exit the protein through transiently opened cavities . Rebinding of CO from the solution to the heme recovers the initial Mb-CO state. This recovery, which takes several milliseconds, has shown to be an intricate reaction with a number of intermediate states. The intermediates were studied with spatially resolved techniques such as crystallography with kinetic resolutions down to the sub-nanosecond time range [2, 3, 11].
To study the rebinding kinetics and the subsequent protein relaxation, step-scan Fourier-transform infrared (FT-IR) spectroscopy with high time resolution was also employed [6, 7, 12]. This method takes advantage from both the sensitivity of IR spectroscopy to electronic changes of the heme cofactor and structural changes of the apo-protein, as well as from the multiplex advantage of FT spectroscopy.
Up to now, most of the applications of step-scan FT-IR spectroscopy to biological samples are restricted to reversible or fast cycling systems. The reason is the necessity to record hundreds of time traces at the various sampling positions of the interferometer to generate the time-resolved data set. It is mandatory that the reaction under study must be precisely synchronised and strictly reversible for the ~1.000 sampling points that constitute the interferogram at a given optical resolution of 4 cm−1. In total, as much as 100.000 repetitions of the same experiment are not uncommon to achieve sufficient signal-to-noise ratio in a time-resolved step-scan experiment of a large protein . This is most practically realised in studies of light-activated proteins by the use of pulsed laser sources [14, 15]. The sample is optically excited and measured and eventually recovers back to the initial state before the next reaction is induced.
For the investigation of irreversible reactions, it is essential that each measurement is performed on a fresh sample of identical concentration. Rödig and Siebert implemented a sample-changing wheel to provide fresh sample after each reaction . Rammelsberg et al. reported a step-scan experiment using an IR microscope and an xy-stage that moves the sample after each excitation . A major problem in both approaches is the change in optical path length when moving from one sampling position to the next because absorption changes as small as <1‰ are recorded on a film thickness of <10 μm! Moreover, both experiments employed hydrated films of membrane protein in which the concentration of compounds is difficult to control.
Here, we present a novel approach to perform step-scan FT-IR experiments for kinetic investigations of irreversible reactions of biological samples in solution. In our setup, the fresh sample condition is provided by continuously pumping the protein solution through a miniaturised flow cell with a sampling volume of only a few nanoliters. The rebinding reaction of CO to myoglobin was studied as a model for the catalytic reaction performed by highly complex heme proteins, like e.g. cytochrome c oxidase. The investigation of the mechanism of the irreversible reaction of cytochrome c oxidase with molecular oxygen was pioneered by Gibson and Greenwood when they developed the so-called flow-flash technique [18, 19]. There, a solution of the CO complex of reduced cytochrome c oxidase was mixed with oxygenated buffer in an observation tube. Heme-bound CO was displaced by a short photolysis flash, molecular oxygen binds to the vacant position and is immediately split . Our experimental setup is designed to finally perform a time-resolved flow-flash IR experiment on the oxygen reaction of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain.
Micro mixer/liquid handling
A 4 mM solution of myoglobin (from horse heart 90%, Sigma) was prepared in 25 mM phosphate buffer at pH 7.0. The solution was placed in an ultrasonic bath for 1 h and centrifuged at 21,900×g (Hettich, Micro 22R, Tuttlingen, Germany). The supernatant was sterile filtrated (0.2 μm cutoff) to remove insoluble components. The resulting myoglobin solution was chemically reduced by the addition of dithionite (final concentration of 16 mM) under anaerobic conditions at a Schlenk line (five cycles of vacuum and purging by Argon). In the final step, Argon was replaced by carbon monoxide, and the resulting solution was stirred for 30 min, yielding a solution of Mb-CO in the presence of an excess of 1 mM non-bound carbon monoxide. The formed Mb-CO complex is stable in solution but photolabile. For UV/Vis spectroscopy, the sample was diluted by 1:100 with 16 mM Na2S2O4 in water to control the formed Mb-CO state of the protein. Shortly before the time-resolved FT-IR experiments were started, the two gastight Hamilton syringes were filled with sample and connected to the inlets of the microfluidic cell under a stream of argon.
Time-resolved FT-IR experiments and data analysis
Step-scan spectroscopy was performed with a spectral resolution of 4.5 cm−1 and a time resolution of 5 μs. Other technical details have been previously described . The FT-IR experiments were performed in two different spectral regions, 2,429–1,669 cm−1 and 1,973–0 cm−1, by using suitable broadband and long-pass interference filters, respectively (E in Fig. 1). Ten time traces were co-added for each mirror position of the interferometer. Each step-scan experiment was repeated four times for the spectral region 2,429–1,669 cm−1 and 15 times for the region 1,973–0 cm−1. At the given flow rate of the myoglobin solution, these acquisition conditions resulted in a total sample consumption of 22.4 μl for measuring the first spectral range 2,429–1,669 cm−1, 469 mirror positions, four averaged full spectral datasets) and 229.5 μl for the second spectral range (1,973–0 cm−1 and 1,024 mirror positions, 15 averaged full spectral datasets). The first 36 spectra of a step-scan experiment, before the laser flash hits the sample, were averaged and used as the reference spectrum to calculate the difference spectra. The resulting difference spectra after photo-activation covered the time range from 5 μs to 2.5 ms. After generation of the matrix of 500 IR difference spectra at the respective time, singular value decomposition (SVD analysis) was applied [13, 23]. Global fit analysis was used to analyse the spectral range of 1,800–1,500 cm−1 as described by Majerus et al. .
Results and discussion
The functionality of proteins is critically dependent on the water content, calling for an aqueous environment. Unlike UV/Vis spectroscopy, the strong absorptivity of water presents a problem in IR spectroscopy of proteins. Thus, an optical path length of a few micrometres is typically used, and hydrated films are often employed to maximise the protein concentration. The challenge of time-resolved IR spectroscopy is to record difference spectra in the presence of the strong background absorption by the aqueous protein solution. IR spectroscopy of proteins in solution suffers from the fact that proteins are difficult to be concentrated to higher than a few millimolar. These considerations set the limits to the design of a microfluidic cell for IR spectroscopy as described in the experimental section. The optical path length of the mixer is 10 μm allowing FT-IR measurements in the spectral regions of water absorptions. The width of the measurement channel of 300 μm compromises adequate transmission of the focussed measuring beam and minimal sample consumption. The sample volume in the measuring spot is exchanged within 23 ms at the chosen flow of 8 μl·min−1. Thus, sample consumption is minimised to about 250 μl of 4 mM protein solution for the entire time-resolved step-scan experiments.
For time-resolved experiments, the photoreaction of Mb-CO was induced by a nanosecond laser pulse to dissociate bound CO from myoglobin. The laser pulse was spatially overlapped with the measuring IR beam (Fig. 1). Absorption changes were recorded with the step-scan technique where the intensity changes at each position of the moving mirror were recorded over time. To follow the kinetics of CO dissociation, the spectral range was limited by an interference filter to the 2,300–1,850 cm−1 range where the C–O stretching vibration of the ligand absorbs. The filter also blocked stray light of the exciting laser from hitting the detector and prevented spectral aliasing. As an additional benefit, undersampling was applied which effectively reduced the number of sampling positions required for a full interferogram.
A detailed view on the early time range (5 to 30 μs in Fig. 9a) shows that the kinetics exhibit a significant deviation from the linear behaviour demonstrating that these structural changes are not correlated to the CO rebinding kinetics. A possible interpretation of this behaviour is an ongoing relaxation of the ligated conformation of myoglobin to deoxy-Mb after photolysis. Step-scan FT-IR experiments on myoglobin from horse-heart in D2O performed by Plunkett et al. agree with our data . These authors found that the decay of the amide I difference band exhibited faster second-order kinetics than the recovery of CO. In contrast, studies on sperm-whale myoglobin in D2O using a tuneable CW lead salt diode infrared probe laser, concluded that protein relaxation after photolysis of CO from Mb-CO is complete after 100 ns . The variation in the experimental results may be attributed to the different behaviour of myoglobin from different organism (horse heart vs. sperm whale) and to the influence of the solvent isotope effect (H2O vs. D2O).
It is demonstrated that the combination of microfluidics with time-resolved step-scan FT-IR spectroscopy can be successfully applied to studies of the reaction kinetics of proteins in solution. As a proof of concept, we studied the kinetics after CO photodissociation from myoglobin. Rebinding of CO from solution to the heme-iron was monitored together with the subsequent protein structural changes. The major technological advance of this novel spectroscopic approach lies in the fact that it allows for step-scan spectroscopy on non-cyclic reactions of proteins in aqueous solution. Previous step-scan studies were commonly performed on hydrated films to reduce the absorption of water in the IR range. We minimised the sample consumption by using a microfluidic flow cell which enables to record multiple co-additions to improve the signal-to-noise ratio while keeping the sample consumption at realistic levels of milligram amounts of protein for a full step-scan experiment. The noise level is reduced by using a dedicated focusing unit which optimises optical throughput of our microfluidic setup.
An additional benefit lies in the opportunity to mix two solutions immediately before excitation. This flow-flash approach has provided valuable insights into the mechanism of the oxygen splitting reaction of cytochrome c oxidase by monitoring the visible absorption of the heme and copper cofactors [18, 19]. Up to now, the short optical path length of ∼10 μm mandatory for mid-IR spectroscopy of aqueous solutions has prevented the application of the classical flow-flash technique to time-resolved IR spectroscopy. As mixing experiments have been successfully performed with the micromixer [21, 29], an oxygen-containing solution will be rapidly mixed with the reduced and CO-bound cytochrome c oxidase, and the irreversible reaction of the enzyme with oxygen is initiated by photodissociation of the carbon monoxide complex. Temporal synchronisation of the flow, photolysis and mirror movement of the interferometer was achieved as demonstrated by the experiments shown here. Thus, the experimental prerequisites are now implemented to perform a flow-flash experiment with FT-IR detection on cytochrome c oxidase in order to study the oxygen-splitting reaction of this central membrane complex of the respiratory chain. These experiments are currently under way.
Financial support for the German part of this research was provided by the Volkswagen Foundation (“Intra- und intermolekulare Elektronenübertragung”). Support for the Austrian part from Carinthian Tech Research AG and the COMET Competence Centre Programme of the Austrian Government is gratefully acknowledged.
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