Vibrational changes associated with CO recombination to ferrous horseradish peroxidase were investigated by rapid-scan FTIR (Fourier-transform IR) spectroscopy in the 1200–2200 cm−1 range. At pH 6.0, two conformers of bound CO are present that appear as negative bands at 1905 and 1934 cm−1 in photolysis spectra. Their recombination rate constants are identical, confirming that they arise from two substates of bound CO that are in rapid thermal equilibrium, rather than from heterogeneous protein sites. A smaller positive band at 2134 cm−1 also appears on photolysis and decays with the same rate constant, indicative of an intraprotein geminate site involved in recombination or, possibly, a weak-affinity surface CO-binding site. Other signals arising from protein and haem in the 1700–1200 cm−1 range can also be time-resolved with similar kinetics.
- carbon monoxide
- Fourier-transform infrared spectroscopy (FTIR spectroscopy)
- horseradish peroxidase
- time-resolved spectroscopy
HRPC (horseradish peroxidase isoenzyme C) catalyses the oxidation of a range of organic compounds by hydrogen peroxide and is a useful model system for studies of redox catalysis involving haem ferryl states. Crystal structures have been solved with various ligands bound and in different stages of its catalytic cycle [1–3]. The protein has a haem B that is proximally ligated to His170. A catalytic pocket is formed at the distal face of the haem involving His42, Arg38 and Phe41. The distal His42 has a pKa of 7.5 , which is shifted to 8.8 when CO is bound to the reduced enzyme . IR [5–7] and Raman  spectra display two conformers of bound CO with υ(CO) vibrations at 1905 and 1934 cm−1 at pH values in the 5–8 range that convert into a single 1934 cm−1 form at high pH. Mutagenesis  and simulation  studies indicate that, in the low-pH form, the bound CO has possible hydrogen-bonding partners of His42 and the guanidinium εNH of Arg38. At high pH, the Nτ of His42 is deprotonated and cannot form a hydrogen bond with the CO, leaving only the guanidinium εNH of Arg38 as a hydrogen-bond donor.
CO binding to HRPC has also been studied by FTIR (Fourier-transform IR) spectroscopy using continuous illumination to generate stationary photodissociated states . In this case, the signal/noise ratio is sufficient to also monitor associated vibrational changes in protein and haem. Such FTIR spectra can be time-resolved by rapid-scan  or step-scan [11,12] techniques. In the present study, rapid-scan FTIR spectroscopy was used to explore further the relation between bound forms of CO in HRPC and to time-resolve the associated vibrational changes of protein and haem during CO recombination.
Time-resolved FTIR spectroscopy
Optimal samples (roughly 12 mM HRPC) had absolute absorption spectra parameters of: (i) an absorption of the 1650 cm−1 peak arising from amide I+water in the 1.0–1.2 range; (ii) a ratio of the maxima at 1650 and 1550 cm−1 between 1 and 1.5, to provide an optimal protein/water ratio; and (iii) ratios of CO/amide II peaks of at least 1/64 (1905/1550 cm−1) and 1/100 (1934/1550 cm−1). The pre-flash level of ferrous–CO compound was 40–50% over the time course of the measurements, with the remainder being the unligated ferrous form.
Spectra were recorded in rapid-scan mode at 275 K on a Bruker ISF66/S spectrometer fitted with a liquid nitrogen-cooled MCT-B detector. Typically, 500 interferograms were averaged to provide an initial dark baseline. Photodissociation of the HRPC ferrous haem–CO compound was achieved with a laser pulse that was synchronized with initiation of recording of 100 single interferograms with a scanner velocity of 280 kHz (time resolution of 16 ms at 4 cm−1 resolution). This cycle was repeated at 3 Hz, and 28000 interferograms were averaged before Fourier transformation to generate a three-dimensional data block of absorbance against frequency against time. The data from three different samples were averaged in order to improve the signal/noise ratio. Difference spectra at specific times after CO photodissociation and kinetics at specific frequencies were extracted from three-dimensional data blocks using Bruker OPUS 6.5 software.
Because of the high protein concentrations required for viable FTIR samples, kinetic data were fitted with a generalized second-order rate equation in which various concentrations of both unligated enzyme and free CO were considered. The averaged pre-flash ratio of (ferrous HRP–CO)/(ferrous HRPC) was estimated from the CO/amide II band ratio and photolysis yield was 40%.
Kinetics of CO rebinding to ferrous HRPC
Figure 1 shows a three-dimensional data block of rapid-scan difference spectra of CO recombination with ferrous HRPC after laser photolysis at 275 K and pH 6.0. The bands correspond closely to those reported previously in photostationary photolysis spectra . Most prominent are the 1934 and 1905 cm−1 troughs that have been assigned to υ(CO) bands of two conformations of haem-bound CO, stabilized by different strengths of hydrogen-bonding interaction(s), either to Arg38 alone (1934 cm−1 conformer) or to both Arg38 and His42 (1905 cm−1 conformer) [6,9]. The decay kinetics of both bands could be fitted with the same second-order rate constant (k) of 1.7±0.1 mM−1·s−1 (Table 1). This confirms that the conformers do not arise from two separate forms of HRPC, but instead are two states that are in rapid thermal equilibrium within the whole HRPC population. Good fits of second-order simulations were achieved by taking a value of 40% photolysis of HRPC–CO (determined from band intensities in dark absolute spectra) to yield 8.8 mM free HRPC and 2.2 mM free CO. Kertesz et al.  and Coletta et al.  measured the second-order rate constant for CO binding to HRPC at 3.4 and 3.0 mM−1·s−1 respectively at pH 7.0 and 293 K. Given that the kinetics are unchanged between pH 5.0 and 7.0 [6,14] and slowed by a factor of 3 upon temperature change from 293 to 275 K , the IR-derived rate constant is roughly in agreement with literature values.
A positive band of lower intensity also appears after photolysis at 2134 cm−1 (Figures 1 and 2). This band, which is evident in earlier work , must arise from an additional bound state of CO that is formed by the photolysed CO. It could be an intraprotein geminate state as is seen, for example, in myoglobin [16,17] and, possibly, other proteins such as NO synthase  and cytochrome ba3  or it could also arise from a weak-affinity surface site that reacts to the increased solution CO concentration after photolysis . The time-resolved spectra of Figures 1 and 2 show that this peak decays with the same rate constant as the bands of haem-bound CO (Table 1). Hence, it can be concluded that the 2134 cm−1 species is already in equilibrium with solution CO in the first (16 ms) trace and must represent a transient site that binds CO before it forms the stable recombination product with ferrous haem or CO binding to a weak-affinity surface site. If a geminate site, a higher occupancy immediately after photolysis might be expected, and this warrants further investigation at smaller time scales by step-scan methods.
Vibrational modes associated with protein and haem
Photolysis/recombination of CO is accompanied by complex vibrational changes in protein and haem that appear below 1800 cm−1 in the ‘fingerprint’ region. Tentative assignments of major bands have been made on the basis of effects of hydrogen/deuterium exchange and comparisons with model materials . Within the available signal/noise levels, all of these bands were found to relax with the same kinetics as the bands of bound CO. For example, the peak/trough at 1548/1539 cm−1, which was assigned to a haem band rather than amide II from its insensitivity to hydrogen/deuterium exchange, had a decay rate constant of 1.7±0.2 mM−1·s−1 and that at 1651−1641 cm−1, which is probably an amide I bandshift, one of 1.6±0.2 mM−1·s−1 (Table 1). Hence, the haem and surrounding protein rearrangements that accompany CO rebinding to haem must occur in synchrony, with no slower post-recombination phases of reorganization.
This rapid-scan time-resolved FTIR spectroscopy study of CO recombination with ferrous HRPC confirms that the two low-pH conformers of CO are thermally interconvertable substates within the whole protein population. It also reveals a synchronous 2134 cm−1 transient that must arise from CO binding to a transient geminate site during rebinding or to a weak-affinity surface site. This work also establishes that the signal/noise ratio is sufficient to extend the kinetic studies into the region below 1800 cm−1 where vibrational changes in the protein and haem cofactor can also be observed to relax in concert with CO recombination. These studies are being extended to smaller time scales with step-scan methods  in order to ascertain geminate states and whether recombination and structural events can be temporally resolved. Progression to application to the functionally more complex cytochrome c oxidase is also underway.
We are grateful to Mr Santiago Garcia for specialist electronic and mechanical support. This work was funded by a grant from the BBSRC (Biotechnology and Biological Sciences Research Council) (BB/C51715X).
Transition Metals in Biochemistry: A joint Biochemical Society meeting with the Inorganic Biochemistry Discussion Group to honour Professor Andrew Thomson FRS, held at University of East Anglia, Norwich, U.K., 24–26 June 2008. Organized and Edited by Steve Chapman (Edinburgh, U.K.), David Richardson (University of East Anglia, U.K.) and Nick Watmough (University of East Anglia, U.K.).
Abbreviations: FTIR, Fourier-transform IR; HRPC, horseradish peroxidase isoenzyme C
- © The Authors Journal compilation © 2008 Biochemical Society