Protein function requires dynamics, the population of and interconversion among multiple conformations and substates of a hierarchical energy landscape. They have been implicated in the activity of cytochrome P450s (CYPs), a superfamily of heme-thiolate enzymes that catalyze oxygen insertion from molecular dioxygen in diverse substrates for a wide range of biosynthetic and metabolic processes (Sigel et al., 2007; Ortiz de Montellano, 2015). For example, crystal structures for a number of CYPs show conformational adaptation for substrate recognition (Williams et al., 2000; Scott et al., 2003; Wester et al., 2003). The most comprehensively studied CYP, P450cam from Pseudomonas putida, undergoes a conformational change from an “open” to a “closed” state upon binding its substrate, camphor (Lee et al., 2010; Asciutto et al., 2011). Whether conformational dynamics play a role in the catalytic cycle beyond substrate recognition is currently a question of high interest (Hollingsworth and Poulos, 2015; Liou et al., 2016, 2017; Batabyal et al., 2017).

CYPs generally undergo a common catalytic cycle (Denisov et al., 2005). Briefly, the binding of substrate to the ferric enzyme displaces a heme-ligated water molecule, leading to conversion of the heme from low to high spin and an increase in redox potential. This promotes a one-electron reduction, followed by binding of O₂. A second one-electron reduction and uptake of two protons generates a highly reactive oxo-ferryl porphyrin radical species, compound I, which is responsible for substrate oxidation. The reduction of CYPs is typically nonspecific and can be mediated by a range of protein and small molecule reductants. However, P450cam is an outlier as the second reduction step has a strict requirement for the native redox partner, the iron-sulfur protein putidaredoxin (Pdx) (Figure 1; Tyson et al., 1972).

A combination of spectroscopic, mutational, thermodynamic, and kinetic binding studies helped to generate the first models of the Pdx complex with P450cam (Sligar et al., 1974; Hintz and Peterson, 1981; Koga et al., 1993; Pochapsky et al., 1994, 1996; Holden et al., 1997; Unno et al., 1997; Kuznetsov et al., 2005, 2006; Zhang et al., 2008; Sevrioukova et al., 2010). A fortuitous observation was made that the mutation, L358P, which removes an interaction of the amide backbone with the thiolate of the cysteine heme ligand, engenders similar spectral changes as binding to Pdx and enables catalysis of camphor hydroxylation with small molecule reductants (Yoshioka et al., 2000, 2002; Tosha et al., 2004). The structure of the L358P P450cam (L358P) subsequently became a potential model for P450cam in the Pdx complex (Nagano et al., 2004; Tosha et al., 2004). Recently, crystal structures have been solved for the Pdx complex (Hiruma et al., 2013; Tripathi et al., 2013). These show a conformational change in P450cam from the closed state of the complex with the camphor substrate to the open state, previously observed in absence of substrate, which reinvigorated interest in the mechanism underlying the effector role of Pdx. However, whether the P450cam adopts an open state in the Pdx complex came into question by an NMR study that indicated a closed state; although, the cyanide complex was under investigation (Skinner et al., 2015). Subsequent double electron-electron resonance (DEER), mutational, and molecular dynamics studies of the unligated enzyme support an open, or partially open, intermediate state (Hollingsworth and Poulos, 2015; Liou et al., 2016, 2017; Batabyal et al., 2017). Complicating the picture is the apparent dependence of the conformational dynamics on the oxidation state of P450cam (Myers et al., 2013; Liou et al., 2016). However, a consensus is forming that Pdx binding indeed promotes conversion to the open state for the ferric enzyme, whereas the ferrous, CO-ligated enzyme appears to remain in the closed state.

The question remains whether a conformational change underlies the effector role of Pdx that is critical to the second reduction step involving the O₂ complex of P450cam. Kinetic studies suggest that the first and second reduction steps involve either different complexes between Pdx and P450cam or different electron transfer pathways (Kuznetsov et al., 2006). Although Pdx does not appear to induce the open state of ferrous CO-ligated P450cam, Raman, infrared (IR), and NMR studies found evidence for spectral perturbation upon Pdx binding (Unno et al., 2002; Nagano et al., 2003; Pochapsky et al., 2003; Tosha et al., 2003; Wei et al., 2005; Asciutto et al., 2009), suggesting that the structure of P450cam could be affected in a way that is connected to the effector role of Pdx in the second reduction step of the catalytic cycle. Toward elucidating the impact of Pdx binding, we applied two-dimensional (2D) IR spectroscopy to the CO-ligated camphor complex of P450cam in the presence and absence of Pdx (subsequently referred to as the Pdx complex and the free wt, respectively), as well as of L358P, the purported model for the P450cam structure in the Pdx complex. The CO vibration served as a reporter of its environment, including the number of distinct environments and the dynamics in each, providing insight into the number of populated conformations and the nature of the energy landscape within them. The time-dependent 2D IR data rigorously demonstrate that the Pdx complex and L358P co-populate two distinct states. For the Pdx complex, the closed state found for the free wt enzyme is the minor species, while a second conformation, which could underlie the effector function, is the dominantly populated state.

The reduced, CO-bound camphor complex of wt P450cam in the absence and presence of Pdx and of L358P first were characterized by linear spectroscopy (Figure 2). For the free wt, a single Gaussian absorption band was found at 1939.4 cm⁻¹, as observed previously by our group and others (O'Keefe et al., 1978; Jung et al., 2002; Basom et al., 2015). In comparison to the free wt, the maxima of the spectra for the Pdx complex and L358P were lower in frequency by ~8 and 4 cm⁻¹, respectively, as reported previously (Yoshioka et al., 2000; Nagano et al., 2003). However, closer inspection revealed asymmetry in the lineshapes for the Pdx complex and L358P, and second derivative spectra indicated the presence of multiple distinct bands (Supplementary Figure 3, Supplementary Table 1). Modeling the spectra as a sum of two Gaussian bands yielded a significantly better fit for both samples (Supplementary Figure 2; Table 1). The frequency of one band was similar to that found for the single band of free wt, differing only by ~2 cm⁻¹, and the frequency of the other band was lower and the same for both the Pdx complex and L358P (1931.2 cm⁻¹; Figure 2; Table 1). For the Pdx complex, the relative integrated absorbance of the band at lower frequency was greater than the one at higher frequency; while for L358P, the opposite was the case.

We next characterized the free wt, L358P, and Pdx complex by 2D IR spectroscopy (Figure 3). 2D IR correlation spectra were generated that connect the initial frequencies of the distribution underlying the absorption bands to the frequencies following a time delay, T_w. Analysis of the 2D spectra provided information about the heterogeneity of frequencies underlying the bands (i.e., inhomogeneous broadening), as well as the dynamics of their interconversion. At the shortest T_w (0.25 ps), the 2D bands were highly elongated along the diagonal (Figure 3, left panels). The spectral elongation reflects high correlation between the initial frequencies (ω₁ axis) and those after the short T_w (ω₃ axis) because the system did not have much time to evolve. As the T_w delay was lengthened, the proteins had increasing time to interconvert among their distribution of states, leading to different final than initial CO frequencies, so the 2D spectra appeared less elongated due to the loss of frequency correlation (e.g., at T_w of 44 ps).

Analysis of the T_w-dependent 2D IR spectra for each sample was performed to obtain the CLS decay, which approximates the inhomogeneous part of the normalized FFCF (Kwak et al., 2007), a quantity that reflects the dynamics of the protein environment coupled to the CO oscillator. The initial value of the CLS provides a measure of the inhomogeneous broadening (i.e., the part of the line width due to frequency heterogeneity from population of a distribution of conformational substates), and the decay of the CLS reflects the timescales over which the inhomogeneous distribution is sampled. The CLS decay and the linear spectrum were co-fit to determine the complete FFCF, which includes the homogeneous contribution, approximately those dynamics that are fast on the IR timescale (Δτ≪1), and deconvolutes the inhomogeneous contribution to provide the variance in frequencies (Δ²) sampled on a particular timescale (τ). Analysis of the 2D IR data for the free wt enzyme yielded an identical FFCF as determined previously (Table 1; Basom et al., 2015). The FFCF shows three components: fast homogeneous dynamics, inhomogeneous dynamics on a ~20 ps timescale, and inhomogeneous dynamics longer than the experimental timescale (~100 ps). The CLS decays determined for the Pdx complex and L358P by analysis around the maximum of the 2D spectra, assuming a single component, are substantially different from each other and from the free wt (Figure 4A). The initial CLS is significantly smaller for the Pdx complex. The CLS decays for the Pdx complex and L358P also reflect much slower dynamics, indicating very slowly interconverting states.

Since the linear spectra for the Pdx complex and L358P were better fit by two component bands, which suggests the population of two distinct conformational states, a more comprehensive analysis of the 2D data was performed. Indeed, closer inspection of the 2D IR data found clear evidence for two component bands. The 2D IR spectrum at T_w of 0.25 ps for the Pdx complex shows obvious asymmetry along the diagonal, with the maximum of absorbance substantially lower in frequency than the center of the entire band (Figure 3). The presence of multiple components was even more apparent from overlay of the diagonal slice through the 2D spectrum with the linear FTIR spectrum (Figure 5A, Supplementary Figure 9). The intensity in a 2D spectrum depends on the fourth power of the transition dipole strength of a vibrational transition, whereas that for the linear spectrum depends on the square, such that vibrational transitions with larger transition dipole strengths are accentuated in a 2D spectrum (Hamm and Zanni, 2011). As a result of this difference, the component band at higher frequency that manifested only as slight asymmetry in the linear spectrum appeared as a clear shoulder band in the 2D diagonal slice. Moreover, comparison of the absorption intensity between the linear and 2D spectrum indicated a ~1.3-fold greater transition dipole strength for the CO vibration associated with the band at higher frequency than the one at lower frequency (Supplementary Table 3).

Further evidence for the presence of multiple components in the spectra for the Pdx complex and L358P is provided by the observation of a shift in the first moment of the diagonal 2D slices to higher frequency with increasing T_w (Figure 5B, Supplementary Table 2). This behavior indicates that the two CO vibrations have distinct vibrational lifetimes in addition to distinct transition dipole strengths. Accordingly, fitting the T_w-dependent amplitude of the diagonal slices yielded lifetimes that are frequency-dependent (Supplementary Figure 8). A global fit to the diagonal spectral data modeling two distinct components gave vibrational lifetimes of 13 ps and 16 ps for the lower and higher frequency bands, respectively. Due to the different lifetimes, the component bands decayed at different rates to result in an apparent shift in the 2D IR spectra to higher frequency with increasing T_w (Figure 3). The effect has become unmistakable by T_w of 44 ps, when the lower frequency component has mostly decayed, resulting in the 2D spectrum showing a maximum at the higher frequency component.

Since the spectra for the Pdx complex and L358P appeared to contain two components, CLS analysis was performed for the T_w-dependent 2D IR data assuming a superposition of two distinct bands, each with a distinct FFCF. The CLS decay for the band at lower frequency was assumed to reflect the FFCF determined for the single absorption band found for the free wt enzyme. With this CLS decay, the relative populations of the two states obtained from the band fitting, and the lifetimes determined from analysis of the time-dependent 2D diagonal spectra, the CLS decay for the second, lower frequency component was extracted from the 2D data for the Pdx complex and L358P. The CLS decays determined for the lower frequency components of the Pdx complex and L358P well-overlapped (Figure 4B), indicating that they reflect the same conformational state with the same energy landscape of substates. Thus, the data were well-modeled by a superposition of the conformation populated by the free wt enzyme and a second state associated with the absorption at lower frequency that is induced by Pdx binding or the L358P mutation. The different FFCFs imply distinct local energy landscapes in the conformations (Figure 6). Compared to the free wt enzyme, the FFCF of the induced state shows that the CO experiences ~30% less inhomogeneous broadening, suggesting lower heterogeneity of states. In addition, the timescale of dynamics measured among inhomogeneous states of 30 ps for the induced state was also slower than the 20 ps measured for the free wt (Table 1). Slower dynamics reflect a more rugged energy landscape, with the 1.5-fold change corresponding to ~4.5 higher energy barriers among substates.

The IR data indicate that the reduced, CO-ligated state of the Pdx complex and L358P populate multiple conformations—the closed state and a second conformation. An earlier report of the linear CO spectra with increasing addition of Pdx noted the asymmetry of the band at high Pdx concentrations, but the feature was attributed to residual unbound P450cam (Nagano et al., 2003). Similarly, the mutation L358P was found to shift the IR absorption of CO to lower frequency (Yoshioka et al., 2000), but the presence of multiple bands was not uncovered. In contrast, the 2D IR data unequivocally report two distinct CO absorptions that provide evidence that the Pdx complex and L358P significantly populate multiple distinct states. That the CO has distinct transition dipole strength in the two states is evident from the different absorption intensity at low and high frequency found for the linear and 2D diagonal spectra (Figure 5A). In addition, the observed shift in the first moment of the 2D spectra with increasing T_w indicates that the CO ligands in the two states have different vibrational lifetimes (Supplementary Figures 7, 8). As expected in this case, at long T_w times the 2D spectra become dominated by the component at higher frequency with the longer vibrational lifetime, which otherwise only manifests as slight asymmetry in the linear IR spectra. Furthermore, the FFCFs that reflect the coupled dynamics of the environment are distinct for the bands (Figure 4B; Table 1), implying different protein motions are experienced by the CO within each state.

In agreement with previous studies (Myers et al., 2013; Liou et al., 2016), the IR data indicate that the Pdx complex of ferrous, CO-ligated P450cam populates a state similar to the closed state observed in crystal structures of the free enzyme, although this state appears to be the minor species. The correspondence of this closed state to the higher frequency CO band is strongly supported by the success in modeling the T_w-dependent 2D data for both the Pdx complex and L358P under the assumption that the FFCF was the same as that for the free wt sample, which is accepted to be in the closed state. Because the FFCF reflects the coupled dynamics of the environment, the observation of equivalent FFCFs means that the CO is sensitive to the same protein motion in the state associated with the high frequency band as in the free wt enzyme. Also in agreement with previous studies (Myers et al., 2013; Liou et al., 2016), the data is not consistent with population of the open state. Previous 2D IR investigation of the CO complex of substrate-free P450cam (Thielges et al., 2011), which adopts the open state (Asciutto et al., 2009; Lee et al., 2010), observed CO absorptions at higher frequencies and determined FFCFs that dramatically differ from those obtained for the Pdx complex. Overall, accounting for the difference in transition dipole strengths, we found that 25% population of the Pdx complex and 54% population of L358P adopt the closed state (Supplementary Table 3).

Although the band at higher frequency is reasonably assigned as the closed state, the small variation observed in the frequencies for the Pdx complex and L358P from the free wt enzyme (lower by 1.9 and 2.6 cm⁻¹, respectively) indicate some difference among the structures. A probable explanation is perturbation of the proximal side of the heme. The mutation L358P removes an electrostatic interaction of the amide backbone of L358 with the thiolate of the cysteine ligand. As suggested previously (Yoshioka et al., 2000), this interaction is likely to withdraw electron density from the heme iron, and ultimately out of a π^* orbital of the CO. Thus, the removal of the amide-thiolate interaction via introduction of L358P is expected to decrease CO bond order and the vibrational frequency. The perturbation however does not appear to affect the FFCF, thus the enzyme motion coupled to the CO vibration.

The question remains what structural change is reflected by the appearance of the second, lower frequency band. One possibility is that the two CO bands are due to co-population of a loosely bound encounter complex and a more tightly associated complex (despite that the P450cam was expected to be >95% bound by Pdx considering the protein concentrations and K_D; Supplementary Material Sample Preparation). Analogous models have been proposed for other redox partners, which in general tend to form dynamic complexes (Skourtis et al., 2010; Bashir et al., 2011; Cruz-Gallardo et al., 2012). A previous study of the Pdx complex employing NMR paramagnetic relaxation enhancement found evidence for the population of a minor state, which was attributed to a tightly bound complex, while the major state was attributed to an encounter complex with P450cam in the closed conformation (Hiruma et al., 2013). In contrast, we find that the 25% populated state, which is associated with the band that exhibits spectral features more similar to the free enzyme, likely corresponds to the closed conformation in the encounter complex. The lower frequency band then is assigned to 75% population of a second, possibly more tightly bound, complex of P450cam and Pdx. In addition, our data indicate that the introduction of the mutation L358P promotes this second state even in the absence of Pdx, leading to its population about equal to the closed state. This interpretation is in line with previous observations that L358P induces spectral perturbations that are similar, but not identical, to those resulting from Pdx binding (Yoshioka et al., 2000, 2002; Tosha et al., 2004).

A potential structural basis for the low frequency band is suggested by the crystal structure of L358P (Nagano et al., 2004). In both wt and L358P structures, the CO ligand is directed toward a groove in helix I, where disruption in hydrogen bonding occurs at Gly248 due to its amide oxygen alternately interacting with the side chain of residue Thr252 (Figure 7; Raag and Poulos, 1989; Nagano et al., 2004). Compared to wt, the L358P structure shows movement of the terminal oxygen of the CO ligand closer to the side chain of Thr252 along with slight displacement of the camphor substrate. These changes are similar to, but less dramatic than, those observed for the O₂ complex, where the hydrogen bond between Thr252 and the backbone of Gly248 is broken, and one forms between Thr252 and the O₂ ligand (Schlichting et al., 2000). The groove in the helix I also widens, and two water molecules enter the pocket. Such changes are thought to facilitate the proton relay network required for O-O bond scission following the second reduction. The possibility that a similar conformational change occurs for CO-ligated P450cam upon Pdx binding is supported by NMR studies, which propose a connection between the changes in the active site to a cis/trans isomerization of Pro89 at a distant location (Asciutto et al., 2009).

Analogous structural changes for the L358P or Pdx complex are likely to result in substantial perturbation to the CO frequency. In particular, the lower frequency observed for L358P and the Pdx complex is consistent with the expected influence of a hydrogen bonding interaction between CO and Thr252 (Spiro and Wasbotten, 2005). Another possibility is supported by the observation from crystallographic study of L358P of electron density for a number of residues that indicate population of multiple conformations (Nagano et al., 2004). Thr252 appears to adopt a second conformation in which side chain rotation places the methyl group instead of the hydroxyl group closer to the CO ligand. The weaker interaction of the methyl group than the polar hydroxyl group with the CO vibration is likely to engender less inhomogeneous broadening, which is consistent with the FFCF determined for the lower frequency component. Further work to model the spectral data using molecular dynamics simulations should enable better understanding of the structural origins.

Significantly, this study provides insight into the perturbation to the conformational ensemble induced by Pdx binding to ferrous, CO-ligated P450cam. We observed that the Pdx complex populates two states that potentially reflect a loosely bound encounter state and a more tightly bound state, in line with our growing understanding of the highly dynamic complexes formed by electron transfer partners (Skourtis et al., 2010; Bashir et al., 2011; Cruz-Gallardo et al., 2012). In comparison to the O₂ complex, the ferrous, CO complex does not bear a partial negative charge on the terminal oxygen of the ligand, but nevertheless reflects a one-electron reduced state with a bound diatomic ligand. The O₂ complex is generally considered a ferric peroxo species but is likely in equilibrium with the ferrous dioxygen species (Denisov et al., 2005). In further studies, 2D IR spectroscopy of a CN⁻ ligand as an IR probe could provide information about the conformational ensemble at the same location in P450cam as CO, but enable comparison to a ferric ligand complex. As in our previous study of camphor binding to P450cam (Basom et al., 2016), incorporation of IR probe groups at amino acid side chains will permit characterization of the effect of Pdx binding at locations in P450cam beyond the distal heme pocket, and potentially enable study of the O₂ complex itself. Nevertheless, the spectral features of the state induced by Pdx binding in this study of the CO-ligated P450cam are consistent with conformational changes that would facilitate subsequent steps in the catalytic cycle found in structural studies (Poulos, 2005), supporting the involvement of the induced state detected in this study in the effector role of Pdx.

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher upon request.

SR performed spectroscopic experiments and sample preparation, analyzed the data, and wrote the paper; EB performed protein expression and purification and edited the paper; MT designed the research, and wrote the paper.