Putidaredoxin-to-cytochrome P450cam electron transfer: differences between the two reductive steps required for catalysis

Cytochrome P450cam (P450cam) is the terminal monooxygenase in a three-component camphor-hydroxylating system from Pseudomonas putida. The reaction cycle requires two distinct electron transfer (ET) processes from the [2Fe-2S] containing putidaredoxin (Pdx) to P450cam. Even though the mechanism of interaction and ET between the two proteins has been under investigation for over 30 years, the second reductive step and the effector role of Pdx are not fully understood. We utilized mutagenesis, kinetic, and computer modeling approaches to better understand differences between the two Pdx-to-P450cam ET events. Our results indicate that interacting residues and the ET pathways in the complexes formed between reduced Pdx (Pdx(r)) and the ferric and ferrous dioxygen-bound forms of P450cam (oxy-P450cam) are different. Pdx Asp38 and Trp106 were found to be key players in both reductive steps. Compared to the wild-type Pdx, the D38A, W106A, and delta106 mutants exhibited considerably higher Kd values for ferric P450cam and retained ca. 20% of the first electron transferring ability. In contrast, the binding affinity of the mutants for oxy-P450cam was not substantially altered while the second ET rates were <1%. On the basis of the kinetic and modeling data we conclude that (i) P450cam-Pdx interaction is highly specific in part because it is guided/controlled by the redox state of both partners; (ii) there are alternative ET routes from Pdx(r) to ferric P450cam and a unique pathway to oxy-P450cam involving Asp38; (iii) Pdx Trp106 is a key structural element that couples the second ET event to product formation possibly via its "push" effect on the heme-binding loop.     

Crystallographic study on the dioxygen complex of wild-type and mutant cytochrome P450cam. Implications for the dioxygen activation mechanism

Two key amino acids, Thr252 and Asp251, are known to be important for dioxygen activation by cytochrome P450cam. We have solved crystal structures of a critical intermediate, the ferrous dioxygen complex (Fe(II)-O2), of the wild-type P450cam and its mutants, D251N and T252A. The wild-type dioxygen complex structure is very much the same as reported previously (Schlichting, I., Berendzen, J., Chu, K., Stock, A. M., Maves, S. A., Benson, D. E., Sweet, R. M., Ringe, D., Petsko, G. A., and Sligar, S. G. (2000) Science 287, 1615-1622) with the exception of higher occupancy and a more ordered structure of the iron-linked dioxygen and two "catalytic" water molecules that form part of a proton relay system to the iron-linked dioxygen. Due to of the altered conformation of the I helix groove these two waters are missing in the D251N dioxygen complex which explains its lower catalytic activity and slower proton transfer to the dioxygen ligand. Similarly, the T252A mutation was expected to disrupt the active site solvent structure leading to hydrogen peroxide formation rather than substrate hydroxylation. Unexpectedly, however, the two "catalytic" waters are retained in the T252A mutant. Based on these findings, we propose that the Thr(252) accepts a hydrogen bond from the hydroperoxy (Fe(III)-OOH) intermediate that promotes the second protonation on the distal oxygen atom, leading to O-O bond cleavage and compound I formation.     

Infrared spectroscopic and mutational studies on putidaredoxin-induced conformational changes in ferrous CO-P450cam

Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has two infrared (IR) CO stretching bands at 1940 and 1932 cm(-1). The former band is dominant (>95% in area) for CO-P450cam free of putidaredoxin (Pdx), while the latter band is dominant (>95% in area) in the complex of CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes a conformational change in the heme active site. To study the mechanism involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR spectroscopic and kinetic analyses of the mutants revealed that an enzyme that has a larger 1932 cm(-1) band area upon Pdx-binding has a larger catalytic activity. Examination of the crystal structures of R109K and R112K suggested that the interaction between the guanidium group of Arg112 and Pdx is important for the conformational change. The mutations did not change a coupling ratio between the hydroxylation product and oxygen consumed. We interpret these findings to mean that the interaction of P450cam with Pdx through Arg112 enhances electron donation from the proximal ligand (Cys357) to the O-O bond of iron-bound O(2) and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby facilitating the O-O bond splitting.     

Activity profile of glutathione-dependent enzymes and respiratory chain complexes in rats supplemented with antioxidants and treated with carcinogens

A real-time optical biosensor study on the interactions between putidaredoxin reductase (PdR), putidaredoxin (Pd), and cytochrome P450cam (P450cam) within the P450cam system was conducted. The binary Pd/P450cam and Pd/PdR complexes were revealed and kinetically characterized. The dominant role of electrostatic interactions in formation of productive electron transfer complexes was demonstrated. It was found that Pd/P450cam complex formation and decay obeys biphasic kinetics in contrast to the monophasic one for complexes formed by other redox partners within the system. Evidence for PdR/P450cam complex formation was obtained. It was found that, in contrast to Pd, which binds only to its redox partners, PdR and P450cam were able to form PdR/PdR and P450cam/P450cam complexes. A ternary PdR/Pd/P450cam complex was also registered. Its lifetime was sufficient to permit up to 60 turnovers to occur. The binding of Pd to P450cam and to PdR within the ternary complex occurred at distinct sites, with Pd serving as a bridge between the two proteins.     

Putidaredoxin-cytochrome p450cam interaction. Spin state of the heme iron modulates putidaredoxin structure

During the monooxygenase reaction catalyzed by cytochrome P450cam (P450cam), a ternary complex of P450cam, reduced putidaredoxin, and d-camphor is formed as an obligatory reaction intermediate. When ligands such as CO, NO, and O2 bind to the heme iron of P450cam in the intermediate complex, the EPR spectrum of reduced putidaredoxin with a characteristic signal at 346 millitesla at 77 K changed into a spectrum having a new signal at 348 millitesla. The experiment with O2 was carried out by employing a mutant P450cam with Asp251 --> Asn or Gly where the rate of electron transfer from putidaredoxin to oxyferrous P450cam is considerably reduced. Such a ligand-induced EPR spectral change of putidaredoxin was also shown in situ in Pseudomonas putida. Mutations introduced into the neighborhood of the iron-sulfur cluster of putidaredoxin revealed that a Ser44 --> Gly mutation mimicked the ligand-induced spectral change of putidaredoxin. Arg109 and Arg112, which are in the putative putidaredoxin binding site of P450cam, were essential for the spectral changes of putidaredoxin in the complex. These results indicate that a change in the P450cam active site that is the consequence of an altered spin state is transmitted to putidaredoxin within the ternary complex and produces a conformational change of the 2Fe-2S active center.     

Complex formation of cytochrome P450cam with Putidaredoxin. Evidence for protein-specific interactions involving the proximal thiolate ligand.

We have performed resonance Raman studies on ferrous NO- and CO-adducts of cytochrome P450(cam) and investigated the effects of diprotein complex formation with reduced putidaredoxin. We have found that the Fe-NO stretching mode of NO-P450(cam) can be resolved into two peaks at 551 and 561 cm(-1), and the binding of putidaredoxin increases the intensity of the high frequency component. Because the Fe-NO mode has been shown to be more sensitive to the nature of the heme proximal ligand than to the distal pocket environment, such a perturbation upon putidaredoxin binding is suggestive of changes in conformation or electronic structure that affect the proximal iron-cysteine bond. In accordance with this idea, the isotope shifts for the Fe-XO stretching and Fe-X-O bending modes (X = N or C) are insensitive to the presence or absence of putidaredoxin, indicating that the geometry of the Fe-X-O unit is not significantly altered by the complex formation. On the other hand, complex formation does induce a perturbation of the low frequency heme vibrational modes, suggesting that alterations of the heme electronic structure and/or geometry take place when putidaredoxin binds. We also find that cytochrome b(5) minimally affects the heme active site of the enzyme, although both putidaredoxin and cytochrome b(5) bind to the same or similar site on P450(cam). These observations suggest that there is a key specific interaction between P450(cam) and putidaredoxin, and that this interaction increases the population of a protein conformation that exhibits structural and/or electronic distortions of the heme group associated with the proximal side of the heme pocket and the S --> Fe electron donation. These electronic and structural changes are potentially correlated with H-bonding to the proximal cysteine.     

Molecular dynamics of the P450cam–Pdx complex reveals complex stability and novel interface contacts

Cytochrome P450cam catalyzes the stereo and regiospecific hydroxylation of camphor to 5‐exo‐hydroxylcamphor. The two electrons for the oxidation of camphor are provided by putidaredoxin (Pdx), a Fe₂S₂ containing protein. Two recent crystal structures of the P450cam–Pdx complex, one solved with the aid of covalent cross‐linking and one without, have provided a structural picture of the redox partner interaction. To study the stability of the complex structure and the minor differences between the recent crystal structures, a 100 nanosecond molecular dynamics (MD) simulation of the cross‐linked structure, mutated in silico to wild type and the linker molecule removed, was performed. The complex was stable over the course of the simulation though conformational changes including the movement of the C helix of P450cam further toward Pdx allowed for the formation of a number of new contacts at the complex interface that remained stable throughout the simulation. While several minor crystal contacts were lost in the simulation, all major contacts that had been experimentally studied previously were maintained. The equilibrated MD structure contained a mixture of contacts resembling both the cross‐linked and noncovalent structures and the newly identified interactions. Finally, the reformation of the P450cam Asp251–Arg186 ion pair in the MD simulation mirrors the ion pair observed in the more promiscuous CYP101D1 and suggests that the Asp251–Arg186 ion pair may be important.     

An anomaly in the resonance Raman spectra of cytochrome P-450cam in the ferrous high-spin state.

Resonance Raman spectra of cytochrome P-450cam (P-450cam) and its enzymatically inactive form (P-420) in various oxidation and spin states were measured for the first time. The Raman spectrum of reduced P-450cam was unusual in the sense that the "oxidation-state marker" appeared at an unexpectedly lower frequency (1346 cm-1) in comparison with those of other reduced hemoproteins (approximately 1355-approximately 1365 cm-1), whereas that of oxidized P-450cam was located at a normal frequency. This anomaly in the Raman spectrum of reduced P-450cam can be explained by assuming electron delocalization from the fifth ligand, presumably a thiolate anion, to the antibonding pi orbital of the porphyrin ring. The corresponding Raman line of reduced P-420 appeared at a normal frequency (1360 cm-1), suggesting a status change or replacement of the fifth ligand upon conversion from P-450cam to P-420. The Raman spectrum of reduced P-450cam-metyrapone complex was very similar to that of ferrous cytochrome b5.     

Resonance Raman detection of bound dioxygen in cytochrome P-450cam

We have used resonance Raman spectroscopy and isotopic labeling techniques to unambiguously assign the dioxygen stretching frequency (vo-o) in the substrate-bound oxygenated complex of cytochrome P-450cam. The frequency found for Vo-o in the P-450cam system (1140 cm-1) is in remarkable agreement with recent studies of thiolate heme model compounds. The general features of the oxy-P-450cam Raman spectra are tabulated and comparisons are made with the oxy complexes of hemoglobin, myoglobin, and various model compounds. Most of the results are qualitatively explained by consideration of electron donation into the pi g (O2)/d pi (M) orbitals of the oxygenated complex (M = Fe or Co). It is also noted that the effect of the "extra" electron in the nitrogen base Co(II) oxy complexes, in some ways, parallels the effect of the lone pair electrons of thiolate in the oxy-P-450cam complex. This is evidenced by the enhanced resonance Raman activity of vo-o in both the Co(II) and P-450 systems as well as by the similarity of the vo-o frequencies.     

Elucidation of the differences between the 430- and 455-nm absorbing forms of P450-isocyanide adducts by resonance Raman spectroscopy.

Alkylisocyanide adducts of microsomal P450 exist in two interconvertible forms, each giving the Soret maximum around 430 or 455 nm. This is demonstrated with a rabbit liver P450 2B4. Resonance Raman spectra of the 430- and 455-nm forms were examined for typical P450s of the two types as well as for P450 2B4 because the 430-nm form of P450 2B4 is liable to change into P420. P450cam and P450nor were selected as a model of the 430- and 455-nm forms, respectively. For the n-butyl isocyanide (CNBu) adduct, the Fe(II)-CNBu stretching band was observed for the first time at 480/467 cm(-1) for P450cam and at 471/459 cm(-1) for P450nor with their (12)CNBu/(13)CNBu derivatives. For P450cam, but not P450nor, other (13)C isotope-sensitive bands were observed at 412/402, 844/835, and 940/926 cm(-1). The C-N stretching mode was identified by Fourier transform IR spectroscopy at 2116/2080 cm(-1) for P450cam and at 2148/2108 cm(-1) for P450nor for the (12)C/(13)C derivatives. These findings suggest that the binding geometry of isocyanide differs between the two forms-bent and linear structures for P450cam-CNBu and P450nor-CNBu, respectively. In contrast, in the ferric state, the Raman (13)C isotopic frequency shifts, and the IR C-N stretching frequencies (2213/2170 and 2215/2172 cm(-1)) were similar between P450cam and P450nor, suggesting similar bent structures for both.     

Revelation of ternary complexes between redox partners in cytochrome P450-containing monooxygenase systems by the optical biosensor method.

Formation of binary and ternary complexes in the water-soluble cytochrome P450cam (P450cam)-containing as well as in the membrane P4502B4(2B4)- and the mixed P450scc-containing monooxygenase systems was investigated in real time by the 'resonant mirror' optical biosensor method. It was shown that the inter-protein electron transfer occurs not only during complex formation but also upon random collision--as was the case with the d-Fp/d-b5 pair (2B4 system). Binary complexes may be either facilitative to electron transfer (electron-transfer complexes) or prohibitive to it (non-productive complexes). Although the binary PdR/Pd and P450cam/Pd complex formation (within the P450cam-system) as well as the binary AdR/Ad and P450scc/Ad complex formation (within the P450scc-system) does occur, the lifetimes of these complexes formed are several orders of magnitude higher than the time required for realization of a complete hydroxylation cycle. At the same time, the lifetimes of the ternary PdR/Pd/P450cam and AdR/Ad/P450scc complexes are sufficient to permit the realization of a complete hydroxylation cycle in either of these systems. For the membrane P450 2B4 system, the formation of both the binary (Fp/2B4 and 2B4/b5) and ternary (Fp/2B4/b5) complexes was registered. The lifetimes of the binary Fp/2B4 and the ternary Fp/2B4/b5 complexes are sufficient for realization of a complete hydroxylation cycle in each of them.     

Atomic force microscopy detection of molecular complexes in multiprotein P450cam containing monooxygenase system

The application of atomic force microscopy (AFM) technique in proteomic research, identification and visualization of individual molecules and molecular complexes within the P450cam containing monooxygenase system was demonstrated. The method distinguishes between the binary protein complexes and appropriate monomeric proteins and, also, between the binary and ternary complexes. The AFM images of the components of a cytochrome P450cam containing monooxygenase system - cytochrome P450cam (P450cam), putidaredoxin (Pd) and putidaredoxin reductase (PdR) - were obtained on a mica support. The molecules of P450cam, Pd and PdR were found to have typical heights of 2.6 +/- 0.3 nm, 2.0 +/- 0.3 and 2.8 +/- 0.3 nm, respectively. The measured heights of the binary Pd/PdR and P450cam/PdR complexes were 4.9 +/- 0.3 nm and 5.1 +/- 0.3 nm, respectively. The binary P450cam/Pd complexes were found to have a typical height of about (3.9 / 5.7 nm) and the ternary PdR/Pd/P450cam complexes, a typical height of about 9.1 +/- 0.3 nm.     

Structural alterations of the heme environment of cytochrome P450cam and the Y96F mutant as deduced by resonance Raman spectroscopy

Resonance Raman spectroscopy at 2.5cm(-1) resolution was used to probe differences in wild-type and Y96F mutant P450cam (CYP101), both with and without bound camphor or styrene substrates. In the substrate-free state, the spin state equilibrium is shifted from 6-coordinate low spin (6CLS) toward more 5-coordinate high spin (5CHS) when tyrosine-96 in the substrate pocket is replaced by phenylalanine. About 25% of substrate-free Y96F mutant is 5CHS as opposed to 8% for substrate-free wild-type P450cam. Spin equilibrium constants calculated from Raman intensities indicate that the driving force for electron transfer from putidaredoxin, the natural redox partner of P450cam, is significantly smaller on styrene binding than for camphor binding. Spectral differences suggest that there is a tilt in camphor toward the pyrrole III ring on Y96F mutation. This finding is consistent with the altered product distribution found for camphor hydroxylation by the Y96F mutant relative to the single enantiomer produced by the wild-type enzyme.     

Effector proteins from P450(cam) and methane monooxygenase: lessons in tuning nature's powerful reagents

Effector proteins alter the kinetic or catalytic course of many oxygenase reactions. One of the first oxygenase effectors to be described was putidaredoxin, which serves to gate electron transfer into oxy-P450(cam). In the nonheme, methane monooxygenase (MMO) system, the B-component (MMOB) serves a distinct effector function by gating substrate and oxygen into the active site of the hydroxylase component (MMOH). Here the binding parameters and binding surfaces of the MMOB-MMOH complex are determined by site-specific labeling, fluorescence titrations, chemical cross-linking, and MALDI-TOF peptide identification. Based on these data, a model for the bimolecular complex is described and a hypothesis for the structural basis for the effector function is elaborated. The bearing on the putidaredoxin effector function is discussed.     

Putidaredoxin-cytochrome P450cam interaction

Cytochrome P450cam (P450cam) catalyzes the monooxygenation of D-camphor. During the enzymatic reaction, oxyferrous, D-camphor-bound P450cam forms a binary complex with reduced putidaredoxin as an obligatory reaction intermediate. We have found that reduced putidaredoxin undergoes EPR-detectable conformational changes upon formation of the intermediate complex and also upon formation of a binary complex with CO- or NO-ferrous, D-camphor-bound P450cam. The structural changes in putidaredoxin are almost identical irrespective of the ligand bound to P450cam, and distinct from and significantly larger than those induced by unliganded ferrous P450cam. The binary complex formation also induce conformational alterations in the CO- and NO-ferrous, D-camphor-bound P450cam, thereby evoking simultaneous changes in the structure of the two proteins. A molecular basis and roles of such structural changes in the D-camphor monooxygenation are discussed.     

A weak Fe-O bond in the oxygenated complex of the nitric-oxide synthase of Staphylococcus aureus

Little is known about the intermediates formed during catalysis by nitric-oxide synthase (NOS). We report here the characterization by resonance Raman spectroscopy of the oxygenated complex of the NOS from Staphylococcus aureus (saNOS) as well as the kinetics of formation and decay of the complex. An oxygenated complex transiently formed after mixing reduced saNOS with oxygen and decayed to the ferric enzyme with kinetics that were dependent on the substrate L-arginine and the cofactor H(4)B. The oxygenated complex displayed a Soret absorption band centered at 430 nm. Resonance Raman spectroscopy revealed that it can be described as a ferric superoxide form (Fe(III)O(2)(-)) with a single nu(O-O) mode at 1135 cm(-1). In the presence of L-arginine, an additional nu(O-O) mode at 1123 cm(-1) was observed, indicating an increased pi back-bonding electron donation to the bound oxygen induced by the substrate. With saNOS, this is the first time that the nu(Fe-O) mode of a NOS has been observed. The low frequency of this mode, at 517 cm(-1), points to an oxygenated complex that differs from that of P450(cam). The electronic structure of the oxygenated complex and the effect of L-arginine are discussed in relation to the kinetic properties of saNOS and other NOS.     

Cytochrome P-450cam and putidaredoxin interaction during electron transfer.

Cytochrome P-450cam, the bacterial hemeprotein which catalyzes the 5-exo-hydroxylation of d-camphor, requires two electrons to activate molecular oxygen for this monooxygenase reaction. These two electrons are transferred to cytochrome P-450cam in two one-electron steps by the physiological reductant, putidaredoxin. The present study of the kinetics of reduction of cytochrome P-450cam by reduced putidaredoxin has shown that the reaction obeys first order kinetics with a rate constant of 33 s-1 at 25 degrees C with respect to: 1) the appearance of the carbon monoxide complex of Fe(II) cytochrome P-450cam; 2) the disappearance of the 645 nm absorbance band of high-spin Fe(III) cytochrome P-450cam; and 3) the disappearance of the g = 1.94 EPR signal of reduced putidaredoxin. This data was interpreted as indicative of the rapid formation of a bimolecular complex between reduced putidaredoxin Fe(III) cytochrome P-450cam. The existence of the complex was first shown indirectly by kinetic analysis and secondly directly by electron paramagnetic resonance spectroscopic analysis of samples which were freeze-quenched approximately 16 ms after mixing. The direct evidence for complex formation was the loss of the EPR signal of Fe(III) cytochrome P-450cam upon formation of the complex while the EPR signal of reduced putidaredoxin decays with the same kinetics as the appearance of Fe(II) cytochrome P-450. The mechanism of the loss of the EPR signal of cytochrome P-450 upon formation of the complex is not apparent at this time but may involve a conformational change of cytochrome P-450cam following complex formation.     

Crystal structures of substrate-free and nitrosyl cytochrome p450cin: implications for o(2) activation

The crystal structure of the P450cin substrate-bound nitric oxide complex and the substrate-free form have been determined revealing a substrate-free structure that adopts an open conformation relative to the substrate-bound structure. The region of the I helix that forms part of the O(2) binding pocket shifts from an α helix in the substrate-free form to a π helix in the substrate-bound form. Unique to P450cin is an active site residue, Asn242, in the I helix that H-bonds with the substrate. In most other P450s this residue is a Thr and plays an important role in O(2) activation by participating in an H-bonding network required for O(2) activation. The π/α I helix transition results in the carbonyl O atom of Gly238 moving in to form an H-bond with the water/hydroxide ligand in the substrate-free form. The corresponding residue, Gly248, in the substrate-free P450cam structure experiences a similar motion. Most significantly, in the oxy-P450cam complex Gly248 adopts a position midway between the substrate-free and -bound states. A comparison between these P450cam and the new P450cin structures provides insights into differences in how the two P450s activate O(2). The structure of P450cin complexed with nitric oxide, a close mimic of the O(2) complex, shows that Gly238 is likely to form tighter interactions with ligands than the corresponding Gly248 in P450cam. Having a close interaction between an H-bond acceptor, the Gly238 carbonyl O atom, and the distal oxygen atom of O(2) will promote protonation and hence further reduction of the oxy complex to the hydroperoxy intermediate resulting in heterolytic cleavage of the peroxide O-O bond and formation of the active ferryl intermediate required for substrate hydroxylation.     

High pressure fourier transform infrared (FT-IR) spectroscopic studies on inducible nitric oxide (NO) synthase active site: a comparison to cytochrome p450CAM.

High pressure Fourier transform infrared (FT-IR) spectroscopy is performed for the first time to analyse the active site of inducible nitric oxide synthase (iNOSox) using the carbon monoxide (CO) heme iron ligand stretch mode (nuCO) as spectroscopic probe. A membrane-driven sapphire anvil high-pressure cell is used. Three major conformational substates exist in substrate-free iNOSox which are characterized by nuCO at approximately 1936, 1945 and 1952 cm(-1). High pressure favors the 1936 cm(-1) substate with a volume difference to the 1945 substate of approximately -21 cm3/mol. The pressure induced cytochrome P420 formation with a reaction volume of approximately -80 cm3/mol is observed. Arginine binding produces a very low nuCO at approximately 1905 cm(-1) caused by the H-bond from the substrate to CO. nuCO for the substates in the substrate-free and arginine-bound proteins shift linearly with pressure which is qualitatively similar to the observation on cytochrome P450cam. The slightly smaller positive slope of the shift in substrate-free iNOSox compared to substrate-free P450cam is interpreted as a slightly lesser compressible heme pocket. In contrast, the significant slower negative slope for arginine-bound iNOSox compared to camphor-bound P450cam results from the different kind of interactions to the CO ligand (electrostatic interaction in P450cam, H-bond in iNOSox).     

Structural stability and dynamics of hydrogenated and perdeuterated cytochrome P450cam (CYP101)

Perdeuterated and hydrogenated cytochrome P450cam (P450cam), from Pseudomonas putida, has been characterized concerning thermal stability and structural dynamics. For the first time, Fourier transform infrared (FTIR) spectroscopy was used to characterize a perdeuterated protein. The secondary structure compositions were determined from the fitted amide I' spectral region, giving band populations at 10 degrees C for the perdeuterated protein of 22% between 1605 and 1624 cm(-1) (beta-sheets), 47% between 1633 and 1650 cm(-1) (alpha-helix (29%) plus unordered/3(10)-helix (18%)), and 28% between 1657 and 1677 cm(-1) (turns) and for the hydrogenated protein of 22% between 1610 and 1635 cm(-1) (beta-sheets), 52% between 1640 and 1658 cm(-1) (alpha-helix (41%) plus unordered/3(10)-helix (11%)), and 24% between 1665 and 1680 cm(-1) (turns).Thermal unfolding experiments revealed that perdeuterated P450cam was less stable than the hydrogenated protein. The midpoint transition temperatures were 60.8 and 64.4 degrees C for the perdeuterated and hydrogenated P450cam, respectively. Step-scan time-resolved FTIR was applied to the P450cam-CO complex to study the ligand-rebinding process after flash photolysis. Rebinding of the ligand occurred with the same kinetics and rate constants k(on), 8.9 x 10(4) and 8.3 x 10(4) M(-1) s(-1) for the perdeuterated and hydrogenated P450cam, respectively.Perdeuterated P450cam was expressed for a neutron crystallographic study to determine the specific hydration states and hydrogen-bonding networks at the active site. The analyses presented here show that perdeuterated P450cam is structurally similar to its hydrogenated counterpart, despite its reduced thermal stability, suggesting that information obtained from the neutron structure will be representative of the normal hydrogenated P450cam.