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Site-specific Characterization of Cytochrome P450cam Conformations by Infrared Spectroscopy Edward John Basom, Micha# Maj, Minhaeng Cho, and Megan C. Thielges Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01520 • Publication Date (Web): 17 May 2016 Downloaded from http://pubs.acs.org on May 19, 2016

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Analytical Chemistry

Site-specific Characterization of Cytochrome P450cam Conformations by Infrared Spectroscopy Edward J. Basom,† Michał Maj,‡§ Minhaeng Cho,‡§ and Megan C. Thielges*,† †Department of Chemistry, Indiana University, 800 East Kirkwood, Bloomington, Indiana 47405, United States ‡Center for Molecular Spectroscopy and Dynamics, Institute for Basic Science (IBS), Seoul 02841, Republic of Korea §Department of Chemistry, Korea University, Seoul 02841, Republic of Korea ABSTRACT: Conformational changes are central to protein function but challenging to characterize with both high spatial and temporal precision. The inherently fast timescale and small chromophores of infrared (IR) spectroscopy are well suited for characterization of potentially rapidly fluctuating environments, and when frequency-resolved probes are incorporated to overcome spectral congestion, enable characterization of specific sites in proteins. We selectively incorporated p-cyanophenylalanine (CNF) as a vibrational probe at five distinct locations in the enzyme cytochrome P450cam and used IR spectroscopy to characterize the environments in substrate and/or ligand complexes reflecting those in the catalytic cycle. MD simulations were performed to provide a structural basis for spectral interpretation. Together the experimental and simulation data suggest that the CN frequencies are sensitive to both long-range influences, resulting from the particular location of a residue within the enzyme, as well as short-range influences from hydrogen bonding and packing interactions. The IR spectra demonstrate that the environments and effects of substrate and/or ligand binding are different at each position probed, and also provide evidence that a single site can experience multiple environments. This study illustrates how IR spectroscopy, when combined with the spectral decongestion and spatial selectivity afforded by CNF incorporation, provides detailed information about protein structural changes that underlie function.

INTRODUCTION Protein function involves conformational changes, ranging from movement of large domains to subtle changes in side chain conformation. However, it remains challenging to characterize these changes with precision sufficient to spatially resolve such structural heterogeneity at the amino acid level and on timescales sufficient to resolve distinct states that interconvert on rapid timescales, such as those involving side chains or water molecules. Infrared (IR) spectroscopy has an inherently fast (sub-ps) timescale and can be applied to small chromophores with approximately local-mode vibrations to generate spatially localized probes of an environment. Moreover, introduction of IR probe groups with spectrally isolated frequencies overcomes the spectral congestion that otherwise hinders IR spectroscopy of macromolecules, and enables application of IR spectroscopy for the characterization of localized, highly fluctuating sites in proteins to elucidate the molecular mechanisms of their function.1,2 We applied IR spectroscopy in combination with site-specific incorporation of p-cyanophenylalanine (CNF) at five individual sites in the enzyme cytochrome P450cam to characterize local changes in distinct microenvironments

throughout its structure in states corresponding to or mimicking those involved in its catalytic cycle. Cytochrome P450s (P450s) are a superfamily of heme (mono)oxygenases that utilize molecular oxygen to catalyze the hydroxylation of hydrocarbons in a wide variety of biological processes, and are particularly important both as central enzymes of drug metabolism and as components of various biotechnological applications.3-5 Cytochrome P450cam (P450cam) from Pseudomonas putida has served as an archetypal P450 for biophysical studies (Figure 1). The catalytic cycle of P450cam involves initial binding of the substrate, camphor, the uptake of two electrons from a protein donor, the binding of molecular oxygen to the heme, followed and substrate hydroxylation. P450cam has traditionally been viewed as a relatively rigid member of the P450 family,6 and has most frequently been studied in a “closed” state. However, a second “open” state of the enzyme adopted in the absence of substrate has recently been detected by both x-ray crystallography and NMR spectroscopy,7,8 raising the possibility that conformational dynamics contribute to its function. Moreover, IR studies have long noted the appearance of multiple vibrational bands for CO when bound to the heme of P450cam in the absence of camphor, providing

*email: [email protected] fax: (812) 855-8300

ACS Paragon Plus Environment

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evidence for multiple unique environments experienced in the active site.9,10 To examine the dynamics of P450cam with increased spatial and temporal resolution, we selectively labeled the enzyme with CNF for use as an IR probe. The aromatic CN provides a relatively intense absorption that can be accurately measured and characterized,11,12 and selective incorporation of CNF is easily accomplished using evolved tRNA/tRNA synthetase pairs that incorporate the modified amino acid at specific positions via amber stop codon suppression.13 Five sites were selected for CNF incorporation (Figure 1). CNF96 and CNF87 are expected to orient the CN probe directly into the active site, whereas CNF98 and CNF201 are progressively distant from the CNF98 CNF96 CNF201

CNF87

CNF305 Figure 1. Chemical structure of CNF and structural model of P450cam (PDB ID: 3L63) showing sites of incorporated CNF. Image generated using UCSF Chimera.

active site. CNF305, selected as a control, is expected to be solvent exposed and distant from the active site. We then employed IR spectroscopy to characterize the CNFlabeled variants in the free state, the complex with camphor, and the complex with camphor and CO (camphor/CO), which reflect or mimic states of its catalytic cycle, as well as in the complex with only CO (in all cases CO substitutes for O2). The number of observed IR absorption bands, their relative intensities, line widths, and frequencies of the CNF probes were analyzed to learn about the protein environment(s) at the labeled residues. We performed all-atom molecular dynamics (MD) simulations and analyzed the trajectories to help elucidate the relationship between the spectral changes and the environment experienced by a CN probe. Together, the experimental and simulation data suggest that the frequency for a particular labeled site is sensitive to the packing of the protein with the CN probe and provide a picture of subtle structural changes at different residues of P450cam during its catalytic cycle.

EXPERIMENTAL SECTION P450cam Expression and Purification. P450cam was expressed from the pDNC334A plasmid kindly provided by Thomas Pochapsky (Brandeis University).14 P450cam was expressed in BL21(DE3) as previously described.15 CNF-labeled P450cam was expressed via the in vivo amber codon suppression method with the pUltraCNF plasmid kindly provided by Peter Schultz (Scripps Research Insti-

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tute).16 Incorporation of CNF at each site was verified by mass spectroscopy of tryptic digests (see Supporting Information). The purified samples showed ASoret/A280 of >1.2, and yields of 3-10 mg/L culture were obtained for all variants. Sample Preparation for FT IR Spectroscopy. To prepare substrate-free P450cam, samples were passed over a 10 cm Sephadex G25 column (GE Life Sciences) equilibrated in 50 mM TrisCl, pH 7.4, with 20% glycerol, then exchanged by three repetitions of ten-fold concentration and dilution into 100 mM potassium phosphate, pH 7, 50 mM KCl, 20% glycerol, and concentrated to 1.5-2.5 mM. The complexes with camphor were prepared in the same buffer but contained 5 mM d-camphor. Based on the determined KDs (see Supporting Information), greater than 99% of the protein for all P450cam variants is expected to be bound in the complex with camphor. P450cam complexes with heme-bound CO were prepared as described previously.15 Briefly, the samples were gently purged under Ar(g) and CO(g) for several minutes, reduced with 15 equivalents of sodium dithionite, and again purged with CO(g). For FT IR spectroscopy, samples were loaded between two CaF2 windows (1 mm thick) with a 38.1 µm Teflon spacer. The visible spectra of all samples in complex with CO were obtained prior to and following data collection (Cary 300, Agilent) to ensure the absence of a peak at 420 nm, which is indicative of the inactive enzyme.17 All samples, regardless of the location of incorporated CNF, displayed the expected band at 450 nm when bound to CO (Figure S6). FT IR Spectroscopy. FT IR spectra were recorded at 2 cm−1 resolution using a dry N2(g)–purged Agilent Cary 670 FT IR spectrometer with a N2(l)-cooled MCT detector. A 4-term Blackman Harris apodization function and zerofilling factor of 8 were applied to process all interferograms, which were averages of 10,000 scans. To generate absorption spectra for each sample, background transmission spectra were collected of wild-type P450cam in 100 mM potassium phosphate, pH 7, with 50 mM KCl, 20% glycerol, and 5 mM d-camphor. The absorption spectra were corrected for a residual slowing varying background absorbance by fitting the regions excluding the bands to a polynomial and its subtraction from the sample spectra. Each background-subtracted absorption spectrum was fit to a Gaussian function or a sum of Gaussian functions (see Supporting Information) to determine the number, relative intensity, frequencies, and line widths of the absorption bands. All reported values are averages from spectra taken of three independent samples. Molecular Dynamics Simulations. Details regarding preparation of the initial P450cam coordinates and parameters for CNF are described in the Supporting Information. MD simulations were performed using AMBER1418 with the ff14SB force field19,20 and TIP3P potential for water molecules.21 The proteins were solvated in a

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cubic (90x90x90 Å3) box in the presence of K+ ions which were added to neutralize the overall charge. Periodic boundary conditions with the Particle Mesh Ewald model22 were used in all calculations with a distance cutoff of 12 Å for non-bonded interactions. All bonds involving hydrogen atoms were constrained via the SHAKE algorithm.23 The system was heated and equilibrated in NVT ensemble for 1 ns, followed by NPT simulation for 500 ps, and another 1 ns NVT step. The production runs were carried out in NVT ensemble with the friction coefficient in the Langevin thermostat set to 0.1 ps−1. The simulations were found to conserve energy, however, minor instabilities were found for the CO-bound proteins resulting in sudden, although not significant, jumps in temperature. This was solved by reducing the time step to 0.5 fs, contrary to 1 fs used in other simulations. MD production runs were collected for 9-14 ns. The trajectory analyses were performed with the aid of CPPTRAJ24 from AmberTools14.

RESULTS AND DISCUSSION Evaluation of Perturbation from CNF Incorporation. Camphor Binding Affinity. P450cam variants were selectively labeled with CNF at five individual residues. To assess the possibility that probe incorporation perturbs camphor recognition, we first determined the affinity with which each variant binds the substrate by spectrophotometric titrations (Figure S7, Table S2). For the wildtype protein, a KD of 1.2 μM was obtained, in agreement with reported literature values (0.8-2.2 μM).25-28 The introduction of CNF305 has no effect on the binding affinity (KD 1.3 μM), as expected, as this residue is distant from the active site, solvent exposed, and was selected as a control. The remaining residues were selected to probe different locations within or surrounding the active site, and in all cases at least minor perturbations to camphor recognition were determined. CNF201 and CNF96 P450cam show slightly larger KDs of 4.1 and 4.7 μM, respectively. Incorporation of CNF98 or CNF87 has a larger impact, but does not abolish binding, increasing the KD about ten-fold (12 and 15 μM, respectively). Visible Spectroscopy. Each P450cam variant was then characterized in each of the four states by visible spectroscopy (Figure S6). As previously observed, the visible spectrum of the wild-type protein shows an intense Soret band at 417 nm in the oxidized free state of the protein, 390 nm in the oxidized complex with camphor,29 and 450 nm30 in the reduced complexes with CO. For both the free state and the CO complexes, no significant differences are apparent among the visible spectra of the CNFlabeled variants and no evidence is found for the inactive P420 state. However, for the complex with camphor, introduction of either CNF96 or CNF98 leads to the appearance of a red-shifted Soret band (~414 nm) that reflects increased content of low spin heme, attributed to displacement of a distally coordinated water molecule31 or conformational changes of the heme porphyrin.32 For

CNF96 the red-shifted band dominates the visible spectrum, whereas for CNF98, the bands at ~390 and 414 nm contribute approximately equally, indicating that CN introduction perturbs the local heme environment in these variants to different extents. Neither of the incorporated nitrile groups could directly displace a heme-bound solvent molecule, but either could indirectly cause solvent displacement or affect the heme conformation. That perturbation occurs only in the camphor-bound state suggests that CNF incorporation at these residues may indirectly force camphor into an orientation which allows for population of the low-spin heme species. It is notable that no perturbation is observed for CNF87, despite that F87 is in van der Waals contact with the substrate. CNF96 and CNF98 are nearer helix I, which forms significant van der Waals contacts with the heme and so could mediate perturbation to the heme conformation. Carbon Monoxide Stretching Modes. We next characterized the CO absorptions of the wild-type and CNFlabeled variants in the CO complexes with and without camphor by FT IR spectroscopy (Figure 2). The spectra were fit to one or a sum of Gaussian functions to determine the number, relative integrated areas, frequencies, and line widths (Table 1, see also the Supporting Information). As observed previously,10,33 the IR spectrum of Table 1. Parameters from Gaussian fits to CO vibrational bands ν (cm-1)

CO CO/camphor CO CO/camphor

CO CO/camphor

CO CO/camphor

CO CO/camphor

CO CO/camphor a b

fwhma (cm-1) CNF87 1940.7 ± 0.1 11.2 ± 0.1 1951.2 ± 0.02 11.0 ± 0.1 1962.2 ± 0.1 11.0 ± 0.1 1940.4 ± 0.03 10.3 ± 0.1 CNF96 1953.3 ± 0.6 22.0 ± 0.3 1964.2 ± 0.1 9.4 ± 0.1 1945.6 ± 0.04 14.4 ± 0.03 1948.9 ± 0.03 7.7 ± 0.2 1964.0 ± 0.1 9.5 ± 0.1 CNF98 1943.2 ± 0.1 20.6 ± 0.2 1961.5 ± 0.2 13.2 ± 0.2 1933.0 ± 1.1 11.5 ± 1.0 1941.3 ± 0.3 12.3 ± 0.3 CNF201 1941.7 ± 0.6 21.1 ± 1.1 1952.9 ± 0.2 10.5 ± 1.3 1962.5 ± 0.2 10.7 ± 0.5 1930.5 ± 0.2 9.0 ± 0.2 1939.5 ± 0.1 13.0 ± 0.1 CNF305 1941.1 ± 0.7 19.9 ± 0.8 1951.7 ± 0.3 11.0 ± 1.4 1962.0 ± 0.6 10.9 ± 1.2 1929.8 ± 0.5 8.3 ± 0.4 1939.7 ± 0.1 12.4 ± 0.2 WT 1939.4 ± 0.4 17.0 ± 0.1 1951.5 ± 0.1 13.4 ± 0.4 1962.7 ± 0.1 9.8 ± 0.1 1939.4 ± 0.2 12.9 ± 0.5

rel. areab (%) 47 ± 1 38 ± 1 15 ± 1

42 ± 1 58 ± 1 82 ± 1 9±1 9±1 81 ± 1 19 ± 1 14 ± 5 86 ± 5 62 ± 5 12 ± 5 26 ± 2 6±1 94 ± 1 60 ± 5 18 ± 8 22 ± 4 5±1 95 ± 1 50 ± 4 33 ± 4 17 ± 1

full width at half the maximum peak height relative integrated area

3


Analytical Chemistry CNF96

CNF98

B

C

D

E

F

G

H

I

J

K

L

1920 1940 1960

1920 1940 1960

1920 1940 1960

1920 1940 1960

CNF201 CNF305

1920 1940 1960

-1

1920 1940 1960

cam/CO

CNF87

A

CO

Norm. Abs.

WT

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Wavenumber (cm )

Figure 2. IR spectra (black lines) showing region of CO stretch vibration for the CO complexes in the absence (AF) and presence (G-L) camphor for wild-type (A,G), CNF87 (B, H), CNF96 (C, I), CNF98 (D, J), CNF201 (E, K), and CNF305 (F, L) P450cam. Component bands from fits to spectra are shown in shaded red, blue, and green.

the wild-type P450cam-CO complex in the absence of camphor shows overlapping absorptions associated with the stretching vibration of the CO that are well fit by a superposition of bands at 1939.5 cm−1, 1951.5 cm−1, and 1963 cm−1 (Figure 2A). Upon formation of the complex with camphor, the spectrum simplifies to a single dominant band at 1939.5 cm−1 (Figure 2G). For all variants, the spectra of the complexes with both camphor and CO are generally similar to that observed for the wild-type protein and show a single dominant CO absorption band (Figure 2G-L), although for all CNFlabeled variants the band is shifted to higher frequency by some extent (1-5 cm−1), and for CNF87 the band is slightly narrower. Additional bands also result from incorporation of CNF at some residues, but in all cases their contribution to the spectrum is very minor. The CO vibrational spectra of the CNF-labeled variants in the absence of camphor show greater differences from CNF incorporation (Figure 2A-F). Consistent with the visible spectral data, the CO vibrations are impacted most greatly by incorporation of CNF96, and to a lesser extent of CNF98. The spectrum of the CNF96 variant is dominated by the band at high frequency (1964 cm−1, where the lowest intensity band appears for the wild-type), shows a second, highly broadened band at 1953 cm−1, and contains no band around 1940 cm−1. For CNF98, the lowest frequency band (1943.2 cm−1) is higher in intensity, and the band at intermediate frequency (1953 cm−1) appears absent, although a minor contribution could be missed due to spectral overlap. The spectra for CNF87, CNF201, and CNF305 P450cam show no significant changes due to CN incorporation, with the exception of a narrower line width found for the lowest frequency band for CNF87. The band at this frequency in the camphor complex is also narrower, suggesting that CNF87 incorporation leads to general restriction of conformation associated with this band. Site-specific characterization of P450cam via Nitrile Stretching Modes. The IR spectra of all proteins with incorporated CNF show absorptions around 2230 cm−1 associated with the stretching vibration of CN (Figure 3 and Table 2).11 In many cases, the spectra are best fit by a superposition of two rather than a single absorption

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band (Tables S3 and S4, see the Supporting Information for a more detailed discussion), and overall, the IR data show that all residues experience two distinct environments in at least one state of P450cam. Additionally, the IR spectra for the different variants vary in the number, frequency, and line widths of the absorption bands. With the exception of CNF305, the frequencies of all nitrile bands depend on the state of P450cam. In fact, the frequency variation observed among the CNF variants in different states (2224.6-2245.0 cm−1) spans the range previously observed for CNF (~2228-2248 cm−1).11-13,34 To provide a structural basis to interpret the spectra, we performed explicit solvent MD simulations of P450cam in the four experimentally characterized states: the free enzyme, the CO complex, the camphor complex, and the camphor/CO complex. We focused this effort on the CNF87 and CNF98 variants, which showed interesting and different changes in the nitrile absorptions upon binding camphor and/or CO compared to CNF305 and CNF201, and we forwent computational investigation of CNF96, which lacks the potential hydrogen bond donor to camphor, as well as most greatly perturbs the visible and CO vibrational spectra. The largest spectral differences among the states of P450cam are observed for CNF87, CNF96, and CNF98, which surround or form the active site. CNF87. For CNF87, the IR spectrum in the free state is best fit by two bands with approximately equal integrated area, suggesting that the CN probe populates two distinct environments about equally. When in complex with only CO, two bands are likewise observed for CNF87, but one dominates (89% relative integrated area). This dominant band is found at the same frequency (2225.5 cm−1) as the lower frequency band observed for the free state. In contrast, for both the camphor and camphor/CO complexes the spectrum is well fit by a single band, suggesting that camphor binding induces structural changes that place the CNF87 side chain into a more homogeneous environment. However, relative to the lower frequency band observed for the free state, the band for the camphor complex is ~1 cm−1 higher in frequency, whereas that for the camphor/CO complex is ~1 cm−1 lower in frequency, implying that differences exist within the local CN environment of the two states. Analyses of the MD trajectories for CNF87 P450cam suggest that the CN participates in a hydrogen bonding interaction with water 45% and 42% of the time in the free state and CO-bound state, respectively, but shows no hydrogen bonding in either the camphor or camphor/CO complexes (Table 3). These relative populations are the same or reasonably close to the 44%, 11%, and zero relative integrated area contributed by the high frequency bands to the spectrum of CNF87 in the free state, CO complex, and complexes with camphor or camphor/CO,

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CNF87

ν (cm-1)

free CO camphor CO/camphor free CO camphor CO/camphor

free CO camphor CO/camphor

free CO camphor CO/camphor a b

56 ± 3 44 ± 3 89 ± 2 11 ± 2

52 ± 8 48 ± 8 42 ± 7 58 ± 7 12 ± 1 88 ± 1

14 ± 3 86 ± 3 18 ± 5 82 ± 5 53 ± 4 47 ± 4 67 ± 2 33 ± 2 45 ± 1 55 ± 1 35 ± 1 65 ± 1 87 ± 1 13 ± 1 87 ± 6 13 ± 6 94 ± 4 6±4 91 ± 4 9±4

full width at half the maximum peak height relative integrated area

Table 3. Frequency of hydrogen bonding to nitriles in MD trajectories CNF87 (%)* CNF98 (%)* free 45.4 2.3 CO 41.7 2.0 camphor 0 0 camphor/CO 0 0 *Cutoff distance = 3 Å; cutoff angle = 135°

respectively. This observation, combined with extensive previous experimental reports of increases in frequency associated with hydrogen bonding to nitriles,35,36 prompts us to assign the higher frequency bands to a hydrogen bonded population of CNF87. The remaining lower frequency bands for the free state and CO complex, as well as the single bands for the camphor and camphor/CO complexes, thus are associated with non-hydrogen bond-

CNF305

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

2250 2220 2235

2250 2220 2235 -1

2250 2220 2235

2220

CNF96

2235 2250 2220 2235

CNF98

camphor cam/CO

CO camphor CO/camphor

rel. areab (%)

CNF201

A

CO

free

fwhma (cm-1) CNF87 2225.5 ± 0.1 8.3 ± 0.2 2230.8 ± 0.1 11.5 ± 0.8 2225.5 ± 0.1 7.4 ± 0.1 2232.1 ± 0.3 8.2 ± 0.9 2226.4 ± 0.01 6.8 ± 0.1 2224.6 ± 0.01 6.3 ± 0.1 CNF96 2230.4 ± 0.3 8.5 ± 0.5 2235.1 ± 0.6 9.8 ± 0.2 2227.2 ± 0.1 5.7 ± 0.2 2232.6 ± 0.4 8.4 ± 0.7 2232.9 ± 0.2 12.1 ± 0.5 2227.8 ± 0.1 6.2 ± 0.1 2235.3 ± 0.1 8.9 ± 0.1 CNF98 2231.6 ± 0.04 8.7 ± 0.1 2229.0 ± 0.4 7.2 ± 0.2 2234.7 ± 0.2 8.5 ± 0.2 2234.6 ± 0.1 10.7 ± 0.3 2230.2 ± 0.6 7.9 ± 0.6 2236.1 ± 0.1 8.0 ± 0.1 CNF201 2228.0 ± 0.1 8.2 ± 0.1 2232.9 ± 0.3 9.9 ± 0.3 2227.7 ± 0.1 8.2 ± 0.1 2233.5 ± 0.2 9.0 ± 0.3 2227.2 ± 0.03 8.0 ± 0.01 2231.2 ± 0.1 11.4 ± 0.1 2227.4 ± 0.03 8.0 ± 0.1 2231.1 ± 0.1 12.6 ± 0.3 CNF305 2235.0 ± 0.1 11.3 ± 0.2 2245.0 ± 0.4 10.6 ± 0.3 2235.1 ± 0.2 10.7 ± 0.2 2244.5 ± 0.5 9.4 ± 1.4 2234.9 ± 0.01 11.2 ± 0.03 2244.3 ± 0.2 8.0 ± 1.4 2234.8 ± 0.1 9.9 ± 0.2 2242.5 ± 0.5 8.6 ± 0.9

Normalized Absorbance

Table 2. Parameters from Gaussian fits to CN vibrational bands

free

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2250

Wavenumber (cm )

Figure 3. IR spectra (black lines) showing region of CN stretch vibration for the free state (A-E), CO complex (F-J), camphor complex (K-O), and camphor/CO complex (P-T) for CNF87 (A, F, K, P), CNF96 (B, G, L, Q), CNF98 (C, H, M, R), CNF201 (D, I, N, S), and CNF305 (E, J, O, T) P450cam. Component bands from fits to spectra are shown in shaded red and blue.

ed CN groups, and the observed variation in their frequencies must be due to another feature of their environments within the protein. Toward understanding the spectral data, we calculated the radial distribution functions (RDFs) for the distances of select sets of atoms to the nitrile nitrogen of CNF87 from the MD trajectories in each of the states (Figure S8). The RDFs represent the relative frequency that the atoms are found at a given distance from the nitrile nitrogen atom during a trajectory. When all atoms (except those of CNF87 itself) are included, the RDFs show spiked amplitude at ~2 Å for the free state and the CO complex, but not for either the camphor or camphor/CO complexes. Likewise, the RDFs for hydrogen or oxygen atoms of water molecules show amplitude at ~2 Å and 3 Å, respectively, for only the free state and CO complex, suggesting that these features reflect the CNF87 when hydrogen bonded to water and are likely associated with the high frequency bands found for these states. To isolate the contributions from structural changes around the non-hydrogen bonded population of CNF87, the RDFs for only the protein non-hydrogen atoms (excluding those of CNF itself) were determined for each of the states. These RDFs each show a local maximum in the amplitude around ~3.5-4.5 Å, followed by a series of maxima at greater distances (Figure S8). We next determined the RDFs for the nitrogen of CNF87 and progressively more selective sets of atoms, as well as analyzed the MD trajectories for atoms that appear with high frequency within progressively larger spheres surrounding the nitrile (Tables S5-S7). For all the states but free CNF87 P450cam, approach of the backbone carbonyl oxygen of Met184 significantly contributes to the shortest distance maxima in the RDFs. In contrast, for the free state, the hydroxyl side chain of Tyr29 is found most frequently within proximity to the nitrile, although they do not ap-

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Analysis of the CNF87 P450cam MD trajectories for geometries conducive to hydrogen bonding finds virtually none involving the nitrile of CNF98 in any of the states (Table 3), indicating that the spectral changes are associated with other factors. As with CNF87, we calculated RDFs for the distances of select sets of atoms to the nitrile nitrogen of CNF98 and analyzed the MD trajectories for atoms that appear with high frequency in proximity to the nitrile (see Supporting Information). When all atoms (except those of CNF98 itself) are included, the RDFs show no amplitude below 2 Å (Figure S8), consistent with the lack of hydrogen bonding interactions. Likewise, little amplitude in the RDFs at short distances was observed for the hydrogen or oxygen atoms of water, although amplitude appeared in the RDFs at greater distances for the free state and CO complex that reflects the increased presence of water in the active site in these states. As for CNF87, we determined the RDFs for all non-hydrogen atoms to the nitrile nitrogen of CNF98 in each state, and local maxima reflecting shortest distance approach of ~3.5-5.5 Å were observed. In contrast to CNF87, the shortest-distance maxima in the RDFs for all nonhydrogen protein atoms were not well accounted for in the RDFs for the oxygen atoms of the protein. Rather, we found that the RDFs for the sulfur on the side chain of Met184 showed high similarity to the shortest-distance maxima observed for all non-hydrogen atoms, and consistently, analysis of the MD trajectories finds close approach of the Met184 side chain to CNF98 in all the states. As for CNF87, we analyzed the MD simulations for each state of CNF98 P450cam to determine RDFs for all nonhydrogen atoms or heteroatoms that approach within 3.55.0 Å of the CNF at least 10% of the time. When only heteroatoms are included, the first moments of the RDFs appear to correlate with the frequencies of the dominant bands (Figure 4), and, interestingly, the plots for the

B

5

3

Distance (Å)

4

5

2232

ρ = 0.95

2228

-1

4 2236

3

ρ = 0.96

2224

CNF98. Unlike the spectra of CNF87, those for all states of CNF98 P450cam show a single dominant band, although in states with bound CO a second minor band (1419% relative integrated area) also appears at lower frequency. The frequency of the dominant band varies significantly among the different states. In the free state, it occurs at relatively low frequency (2231.6 cm−1), whereas upon binding either CO or camphor individually, the band shifts to higher frequency by ~3 cm−1, and upon binding both ligands, is 4.5 cm−1 higher in frequency. Thus, like CNF87, CNF98 experiences different environments in the different states of P450cam.

A

Center Frequency (cm )

pear to form a hydrogen bond. Interestingly, the proximity of protein non-hydrogen atoms or heteroatoms to the nitrile appears to correlate with the frequency of the bands (Figure S9). Consistent with this observation, we determined the RDFs for all non-hydrogen atoms or heteroatoms that approached within 3.5-5.0 Å of the nitrile nitrogen greater than 10% of the time, and plots of the frequencies of the IR bands against the first moments of the RDFs show high correlation (Figures 4 & S9).

RDF

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3

3.4

3.8

4.2

4.6

5

RDF First Moment (Å) Figure 4. RDFs centered at the nitrile nitrogen on CNF87 (A) and CNF98 (B) for closely approaching heteroatoms (within 4 Å of the nitrile nitrogen for more than 10% of the time) in the free state (black), CO complex (red), camphor complex (blue), and camphor/CO complex (green). (C) Plot of frequency of the IR bands against the first moment of the RDFs for closely approaching heteroatoms for each examined state of CNF87 (black) and CNF98 (red) P450cam. For CNF87, which showed two bands of about equal intensity, the data for the band at higher frequency assigned to the state hydrogen bonded to water and correlated with the RDF for all water oxygen atoms with a 3 Å cutoff is shown as a diamond symbol. The Pearson correlation coefficient is given next to each data set, and a fit to a line is provided to guide the eye.

CNF87 and CNF98 data show approximately the same slope, consistent with the similar mechanism underlying the spectral changes. A similar correlation is not observed for CNF98, however, when all non-hydrogen atoms are included in the analysis (Figure S9), which is consistent with the solvatochromic shifts reflecting the influence of repulsive interactions from packing closely against heteroatoms. The plots of the data for CNF87 and CNF98 however are offset, indicating that, while the frequency is sensitive to the proximity of protein heteroatoms, the frequency variation at each residue arising from the short-range interactions is superimposed on a longerrange solvatochromic-induced shift. For example, the nitrile of CNF87 is located further from the surface of P450cam than is that of CNF98, and a dielectric shielding effect of the protein could be the origin of the linear plots’ offset. This would also explain why the frequency of ~2231 cm−1 assigned to the hydrogen-bonded nitrile of CNF87 in the water-filled active site of P450cam is unexpectedly low on an absolute scale compared to the frequency of 2235 cm−1 for CNF in bulk water, despite that notably, the ~5-6 cm−1 differences between the two bands observed in the free state and CO complex are consistent with hydrogen bond formation35,36 and that the distances and geome-

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tries from the MD simulations suggest formation of moderately strong hydrogen bonds.37 The varying frequencies of the IR bands report different environments for CNF87 and CNF98 among the states, providing localized information about the conformational changes evoked previously in structural and spectroscopic studies of P450cam. In agreement with previous studies,7,38 our combined IR data and MD simulations suggest that CNF87 and CNF98 report on the varying presence of water in the active site and/or changes in nearby helices F and G during the transition from the “open” state in the substrate-free enzyme to the “closed” state upon binding camphor (Figure 5A). For example, the presence of the high frequency bands attributed to hydrogen bonding with water in the free state and CO complex suggest that CNF87 experiences a solvent-filled active site whenever camphor is absent. That the contribution of the high frequency band is lower for the complex with CO implies that CO binding destabilizes the water hydrogen bonded to CNF87 and/or leads to partial active site desolvation. We note that this IR data helps clarify an apparent discrepancy in the estimation of greater or lower water occupancy between x-ray and NMR studies that characterized the free and CO-bound states, respectively.7,8 The frequencies of the IR bands attributed to the nonhydrogen bonded population of CNF87 and those for CNF98 appear to reflect their high sensitivity to the proximity of helices F and G, and to some extent other structural differences among the states. In all the ligand complexes, CNF87 most frequently approaches the backbone carbonyl oxygen of Met184 at end of helix F, whereas uniquely in the free state it most frequently approaches the side chain of Tyr29. Similarly, the side chain of CNF98 points toward helices F and G, but it comes into closer contact with the side chain rather than backbone of Met184. Comparison of the differences in frequencies between the free state and camphor complex, which reflects the first step of the native P450cam catalytic cycle, suggests that both the CNF87 and CNF98 side chains become more highly packed against helix F upon camphor binding. In contrast, the data suggests that the side chain of CNF87 increases its distance from helix F upon binding of CO to form the camphor/CO complex, whereas in this process CNF98 approaches helix F more closely. The different behavior for CNF87 and CNF98 indicates that in addition to “open” and “closed” states of the free state and camphor complex, distinct structural changes occur in P450cam upon forming the camphor/CO complex. Together the IR data for CNF98 and CNF87 data provide evidence for local variation in P450cam structure as it proceeds through each of the states mimicking its catalytic cycle. Finally, it is interesting that the IR spectra for CNF98 show a small lower frequency band that reports on a small population in a distinct environment whenever CO is bound. Given that CNF98 is found by MD simulations to be inaccessible to water, the minor bands likely reflect a distinct environment arising from the protein

A

helix F FG loop

camphor helix G

B

Y29

heme

C M184

M184 CNF98 CNF87

Figure 5. Average structures of CNF98 P450cam (A) from MD trajectories of the free state (purple) and camphor complex (green) highlighting the F and G helices, FG loop, heme, and camphor. Close up of the average structures from MD simulations of the free state (purple) and camphor complex (green) showing local environments around CNF87 (B) and CNF98 (C). Amino acids are specified with single letter codes.

structure. Notably, the RDFs for the closely approaching heteroatoms appear bimodal for the states with bound CO (Figure 4B). CNF96. Although we did not perform MD simulations for all the CNF-labeled variants, our analysis of CNF87 and CNF98 guides spectral interpretation for the other residues. The spectral changes for CNF96 among the different states show greater complexity, as expected, since previous structural studies find the side chain of Tyr96 oriented directly into the active site pocket where it acts as a hydrogen bond donor to water or the bound camphor substrate.29,39,40 For the free state and the complex with CO, the IR spectra show two bands with relative intensities that report two distinct environments experienced about equally by CNF96 in the absence of camphor (Figure 3 and Table 2). As with CNF87, which is also oriented into the active site, the bands for CNF96 at higher and lower frequency likely reflect the presence and absence, respectively, of a hydrogen bond to the nitrile in the solvent-filled active site of these states. Analyses of the MD simulations for CNF87 and CNF98 in the absence of camphor find that the hydroxyl group of Tyr96 acts as a hydrogen bond donor to active site water during 83% of the four trajectories, but, interestingly, also 68% of the time acts as a hydrogen bond acceptor to the hydroxyl group of Thr101 (Figure S10). CNF96 maintains the ability to participate in this interaction with Thr101, and although no longer can serve as a hydrogen bond donor, could alternately act as hydrogen bond acceptor to water in the active site. In contrast to the spectra without camphor, the

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spectrum for the camphor complex shows a single band, although the band is relatively broad, suggesting greater heterogeneity within the state. Single bands are also observed for CNF87 and CNF98 in the camphor complex, and thus the three residues closest to the active site collectively experience single distinct environments in the complex with camphor. In the camphor/CO complex, the spectrum for CNF96 is dominated by a single band (88% relative integrated area), with a minor band appearing at lower frequency. The frequency of the dominant band is relatively high (2235 cm−1), consistent with a tightly packed side chain in the tertiary complex. Potentially the CN group of CNF96 is sensitive to the carbonyl moiety of camphor to which Tyr96 forms a hydrogen bond in the native complex. Finally, it is also interesting that for the CO complexes with and without camphor, the integrated areas of the CN bands at ~2227 cm−1 and 2232 cm−1 for the variants containing CNF96 and CNF87, respectively, appear to approximately correspond and co-vary with the relative integrated areas of the CO band at 1963 cm−1 (Tables 1 and 2), which has been previously attributed to interaction of the CO with active-site water.10,33 This demonstrates that the two independent IR probes, CN and CO, report consistent relative populations of distinct environments at the same location in P450cam. CNF201 and CNF305. Compared to the variants with CNF introduced immediately within or next to the active site, the spectra of CNF305 and CN201 show less sensitivity to the binding of CO or camphor ligands. For CNF305, the spectra contain a dominant band at 2235 cm−1 and a very minor, but reproducible, band at higher frequency (~2244.5 cm−1) in all states examined. That the frequency of the dominant bands are the same as for CNF in bulk water and are independent of ligand binding reflect that CNF305 is located at the protein surface distant from the active site. For CNF201, the spectra in all states are best fit by two bands with similar relative intensities indicating the existence of two substantial populations of CNF201 in which the CN experiences distinct environments. No water molecules are found to hydrogen bond with the Tyr201 side chain in the simulations of the CNF87 and CNF98 proteins, so the two distinct environments associated with the two bands for CNF201 likely arise from the protein structure. The influence of close heteroatom contact gleaned from our analysis of the CNF87 and CNF98 data suggests that the side chain of CNF201 is more tightly packed in one environment than in the other. CNF201 is also near Lys197, and fluctuations that vary the CNF proximity to the positively charged side chain might underlie the multiple IR bands. Nevertheless, small changes in the relative areas of the bands are found among the states of P450cam, indicating that CNF201 is variably stabilized in the two environments in the different complexes with CO and/or camphor to some extent. In addition, a small (~1 cm−1) decrease in the frequencies of both bands is ob-

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served in the complexes with camphor, consistent with compaction of the structure around CNF201 upon camphor binding.

CONCLUSIONS This study illustrates selective incorporation of CNF vibrational probes combined with FT IR spectroscopy to enable characterization of the environments at five distinct locations in P450cam for states reflective of the catalytic cycle. Analysis of the IR data in combination with the MD simulations suggests that the CN frequencies are sensitive to both long-range influences, resulting from the particular location of a residue within the enzyme, as well as short-range influences from hydrogen bonding and packing interactions. Hydrogen bonding appears to substantially impact the CN vibrations and lead to large increases in frequency. When not hydrogen bonded, variation in CN frequency appears to reflect sensitivity to local changes in its packing, particularly against heteroatoms of the protein. While significant effort has been directed towards developing rigorous descriptions of the physics underlying vibrational shifts of CNF probes incorporated within proteins, such as MD simulations combined with electrostatic calculations and a linear vibrational Stark model, such approaches may be insufficient to capture the influence of specific, localized interactions such as hydrogen bonding. A distributed site model has been developed that more accurately estimates vibrational frequencies, but requires relatively intensive computational effort.41,42 Additionally, more comprehensive, but similarly computationally intense, treatments of vibrational frequency shifts find that inductive, repulsive, and dispersive intermolecular interactions also potentially contribute, at least for amide vibrations.43,44 In the future, analysis of all interaction energy contributions to the CN probe frequencies will undoubtedly shed more light on the origin of the observed vibrational frequencies of the CNF probes in P450cam. Other studies have alternately taken experimental approaches to unravel the contribution of hydrogen bonding to CN spectral changes. For example, correlation of IR frequencies with 13C NMR chemical shifts has been used to guide interpretation,36,45 however this approach requires introduction of isotopically labeled CN. More recently, temperature-dependent studies of CN absorptions were demonstrated to unravel the contribution of hydrogen bonding,46 but the approach is not amenable to proteins susceptible to heat-induced denaturation and inactivation, such as P450s and other enzymes. As an alternative, our current study suggests that the IR spectral changes of CN at particular location may be at least qualitatively interpreted by simple analysis of MD simulations. In some cases, the IR spectra provide evidence for multiple distinct environments experienced by side chains in a single state of P450cam, due to either hydrogen bonding to active site water (CNF87) or to different protein environments (CNF98). In general, those CNF residues placed closer to the active site pocket report greater dif-

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ferences among their spectra upon formation of complexes with the camphor and/or CO ligands. Specifically, the spectral differences among the states reflecting the P450cam catalytic cycle suggest changes in P450cam involving helix F that result in its variable packing against the CNF87 and CNF98 side chains. Importantly, the study illustrates how the fast timescale of IR spectroscopy, combined with spectral decongestion and spatial selectivity afforded by incorporation of CNF, enables resolution of rapidly exchanging states, such as those differentiated by water hydrogen bonding, and at localized sites throughout proteins to provide detailed information about protein structural changes that underlie function.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS The authors thank Thomas Pochapsky at Brandeis University for providing the pDNC334A plasmid and Peter Schultz at the Scripps Research Institute for providing the pUltraCNF plasmid. The authors also acknowledge D. Matelska and B. Błasiak for helpful discussions. This work was supported by Indiana University. E.J.B. was also supported by the Graduate Training Program in Quantitative and Chemical Biology (T32 GM109825). M.C. thanks the financial support by the Institute for Basic Science (IBS-R023-D1).

ASSOCIATED CONTENT Supporting Information Procedural details and additional data from mass spectrometry, visible absorption spectroscopy, determination of binding affinities, evaluation of fits to IR spectra, and analysis of MD simulations, as well as oligonucleotides for plasmid preparation (PDF). The Supporting Information is available free of charge on the ACS Publications website.

REFERENCES (1) Kim, H.; Cho, M. Chem. Rev. 2013, 113, 5817-5847. (2) Chin, J. K.; Jimmenez, R.; Romesberg, F. E. J. Am. Chem. Soc. 2001, 123, 2426-2427. (3) Denisov, I. G.; Makris, T. M.; Sligar, S. G.; Schlichting, I. Chem. Rev. 2005, 105, 2253-2277. (4) Bernhardt, R. J. Biotechnol. 2006, 124, 128-145. (5) Poulos, T. L. Chem. Rev. 2014, 114, 3919-62. (6) Winn, P. J.; Ludemann, S. K.; Gauges, R.; Lounnas, V.; Wade, R. C. Proc. Natl. Acad. Sci. USA 2002, 99, 5361-5366. (7) Lee, Y. T.; Wilson, R. F.; Rupniewski, I.; Goodin, D. B. Biochemistry 2010, 49, 3412-3419. (8) Asciutto, E. K.; Young, M. J.; Madura, J.; Pochapsky, S. S.; Pochapsky, T. C. Biochemistry 2012, 51, 3383-93. (9) O'Keefe, D. H.; Ebel, R. E.; Peterson, J. a.; Maxwell, J. C.; Caughey, W. S. Biochemistry 1978, 17, 5845-5852. (10) Jung, C.; Hoa, G. H.; Schröder, K. L.; Simon, M.; Doucet, J. P. Biochemistry 1992, 31, 12855-12862. (11) Getahun, Z.; Huang, C.-Y.; Wang, T.; De Leon, B.; DeGrado, W. F.; Gai, F. J. Am. Chem. Soc. 2003, 125, 405-411.

(12) Horness, R. H.; Basom, E. J.; Thielges, M. C. Anal. Methods 2015, 7, 7234-7241. (13) Schultz, K. C.; Supekova, S.; Ryu, Y.; Xie, J.; Perera, R.; Schultz, P. G. J. Am. Chem. Soc. 2006, 128, 13984-13985. (14) Ouyang, B.; Pochapsky, S. S.; Pagani, G. M.; Pochapsky, T. C. Biochemistry 2006, 45, 14379-14388. (15) Basom, E. J.; Spearman, J. W.; Thielges, M. C. J. Phys. Chem. B 2015, 119, 6620-6627. (16) Chatterjee, A.; Sun, S. B.; Furman, J. L.; Xiao, H.; Schultz, P. G. Biochemistry 2013, 52, 1828-1837. (17) Yu, C.-A.; Gunsalus, I. C. J. Biol. Chem. 1972, 249, 102-106. (18) Case, D. A. et al., AMBER 2014. University of California, San Francisco, 2014. (19) Price, D. J.; Brooks, C. L., III J. Chem. Phys. 2004, 121, 10096-103. (20) Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling, C. J. Chem. Theory Comput. 2015, 11, 3696713. (21) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. J. Chem. Phys. 1983, 79, 926-935. (22) Darden, T.; York, D.; Pedersen, L. J. Chem. Phys. 1993, 98, 1008910092. (23) Lambrakos, S. G.; Boris, J. P.; Oran, E. S.; Chandrasekhar, I.; Nagumo, M. J. Comput. Phys. 1989, 85, 473-486. (24) Roe, D. R.; Cheatham, T. E., III J. Chem. Theory Comput. 2013, 9, 3084-95. (25) Schulze, H.; Hui Bon Hoa, G.; Jung, C. Biochim. Biophys. Acta 1997, 1338, 77-92. (26) Atkins, W. M.; Sligar, S. G. J. Biol. Chem. 1988, 263, 18842-18849. (27) Deprez, E.; Di Primo, C.; Hui Bon Hoa, G.; Douzou, P. FEBS Letters 1994, 347, 207-210. (28) Di Primo, C.; Hui Bon Hoa, G.; Douzou, P.; Sligar, S. J. Biol. Chem. 1990, 265, 5361-5363. (29) Poulos, T. L.; Raag, R. FASEB J. 1992, 6, 674-679. (30) Peterson, J. A.; Griffin, B. W. Arch. Biochem. Biophys. 1972, 151, 427-433. (31) Poulos, T. L.; Cupp-Vickery, J.; Li, H., In Cytochrome P450: Structure, Mechanism, and Biochemistry, Ortiz de Montellano, P., Ed. Springer: New York, 1995; pp 125-150. (32) Colthart, A. M.; Tietz, D. R.; Ni, Y.; Friedman, J. L.; Dang, M.; Pochapsky, T. C. Sci. Rep. 2016, 6, 22035. (33) Jung, C.; Ristau, O.; Schulze, H.; Sligar, S. G. Eur. J. Biochem. 1995, 235, 660-669. (34) Shrestha, R.; Cardenas, A. E.; Elber, R.; Webb, L. J. J. Phys. Chem. B 2015, 119, 2869-2876. (35) Aschaffenburg, D. J.; Moog, R. S. J. Phys. Chem. B 2009, 113, 12736-12743. (36) Fafarman, A. T.; Sigala, P. A.; Herschlag, D.; Boxer, S. G. J. Am. Chem. Soc. 2010, 132, 12811-12813. (37) Jeffrey, G., An Introduction to Hydrogen Bonding. Oxford University Press: Oxford, 1997. (38) Skinner, S. P.; Liu, W.-M.; Hiruma, Y.; Timmer, M.; Blok, A.; Hass, M. A. S.; Ubbink, M. Proc. Natl. Acad. Sci. 2015, 112, 9022–9027. (39) Poulos, T. L.; Finzel, B. C.; Howard, A. J. J. Mol. Biol. 1987, 195, 687-700. (40) Raag, R.; Poulos, T. L. Biochemistry 1991, 30, 2674-2684. (41) Choi, J. H.; Cho, M. J. Chem. Phys. 2011, 134, 154513. (42) Lee, H.; Choi, J. H.; Cho, M. J. Chem. Phys. 2012, 137, 114307. (43) Błasiak, B.; Cho, M. J. Chem. Phys. 2014, 140, 164107. (44) Błasiak, B.; Cho, M. J. Chem. Phys. 2015, 143, 164111. (45) Bagchi, S.; Fried, S. D.; Boxer, S. G. J. Am. Chem. Soc. 2012, 134, 10373-10376. (46) Adhikary, R.; Zimmermann, J.; Dawson, P. E.; Romesberg, F. E. Anal. Chem. 2015, 87, 11561-7.

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Analytical Chemistry for TOC only: -1

Center Frequency (cm )

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free

bound

CNF98 CNF87 RDF First Moment (Å)

IR spectra


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Site-Specific Characterization of Cytochrome ... - ACS Publications

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