Conformational changes in cytochrome c and cytochrome oxidase upon complex formation: a resonance Raman study

The fully oxidized complex of cytochrome c and cytochrome oxidase formed at low ionic strength was studied by resonance Raman spectroscopy. The spectra of the complex and of the individual components were compared over a wide frequency range using Soret band excitation. In both partners of the complex, structural changes occur in the heme groups and in their immediate protein environment. The spectra of the complex in the 1600-1700 cm-1 frequency range were dominated by bands from the cytochrome oxidase component, whereas those in the 300-500 cm-1 range were dominated by bands from the cytochrome c component, hence allowing separation of the contributions from the two individual species. For cytochrome c, spectral changes were observed which correspond to the induction of the conformational state I and the six-coordinated low-spin configuration of state II on binding to cytochrome oxidase. While in state I the structure of cytochrome c is essentially the same as in solution, state II is characterized by a structural rearrangement of the heme pocket, leading to a weakening of the axial iron-methionine bond and an opening of the heme crevice which is situated in the center of the binding domain for cytochrome oxidase. The relative contributions of the two cytochrome c states were estimated to be approximately in the ratio 1:1 in the complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Replacements in a conserved leucine cluster in the hydrophobic heme pocket of cytochrome c.

A cluster of highly conserved leucine side chains from residues 9, 68, 85, 94, and 98 is located in the hydrophobic heme pocket of cytochrome c. The contributions of two of these, Leu 85 and Leu 94, have been studied using a protein structure-function-mutagenesis approach to probe their roles in the maintenance of overall structural integrity and electron transfer activity. Structural studies of the L85C, L85F, L85M, and L94S mutant proteins show that, in each case, the overall fold of cytochrome c is retained, but that localized conformational shifts are required to accommodate the introduced side chains. In particular, the side chains of Cys 85 and Phe 85 form energetically favorable interactions with Phe 82, whereas Met 85 takes on a more remote conformation to prevent an unfavorable interaction with the phenyl ring of Phe 82. In the case of the L94S mutant protein, the new polar group introduced is found to form hydrogen bonds to nearby carbonyl groups. In all proteins with substitutions at Leu 85, the hydrophobic nature of the heme pocket is preserved and no significant decrease in heme reduction potential is observed. Despite earlier predictions that Leu 85 is an important determinant in cytochrome c electron transfer partner complexation, our studies show this is unlikely to be the case because the considerable surface contour perturbations made by substitutions at this residue do not correspondingly translate into significant changes in electron transfer rates. For the L94S mutant protein, the substitution of a polar hydroxyl group directly into the hydrophobic heme pocket has a larger effect on heme reduction potential, but this is mitigated by two factors. First, the side chain of Ser 94 is rotated away from the heme group and, second, the side chain of Leu 98 shifts into a portion of the new space available, partially shielding the heme group. The Leu 94 Ser substitution does not perturb the highly conserved interface formed by the nearly perpendicular packing of the N- and C-terminal helices of cytochrome c, ruling this out as the cause of this mutant protein becoming thermally labile and having a lower functional activity. Our results show these effects are most likely attributable to disruption of the heme pocket region. Much of the ability of cytochrome c to absorb the introduction of mutations at Leu 85 and Leu 94 appears to be a consequence of the conformational flexibility afforded by the leucine cluster in this region as well as the presence of a nearby internal cavity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural changes in cytochrome c oxidase induced by cytochrome c binding. A resonance raman study

Electrostatically stabilized complexes of fully oxidized cytochrome c oxidase from Paracoccus denitrificans and horse heart cytochrome c were studied by resonance Raman spectroscopy. The experiments were carried out with the wild-type oxidase and a variant in which a negatively charged amino acid in the binding domain (D257) is replaced by an asparagine. It is shown that cytochrome c induces structural changes at heme a and heme a(3) which are reminiscent to those found in mammalian cytochrome c oxidase-cytochrome c complex. The spectral changes are attributed to subtle changes in the heme-protein interactions implying that there is a structural communication from the binding domain even to the remote catalytic center. Only for the heme a modes minor spectral differences were found in the response of the wild-type and the D257N variant oxidase upon cytochrome c binding indicating that electrostatic interactions of aspartate 257 are not crucial for the perturbation of the catalytic site structure in the complex. On the other hand, in none of the complexes, structural changes were detected in the bound cytochrome c. These findings are in contrast to previous results obtained with beef heart cytochrome c oxidase which triggers the formation of a new conformational state of cytochrome c assumed to be involved in the biological electron transfer process.     

A polypeptide chain-refolding event occurs in the Gly82 variant of yeast iso-1-cytochrome c

The replacement of Phe82 in yeast iso-1-cytochrome c by a glycine residue substantially alters both the tertiary structure and electron transfer properties of this protein. The largest structural change involves a polypeptide chain refolding of residues 79 through 85. Refolding places glycines 82, 83 and 84 immediately adjacent to the plane of the heme group in a spatial positioning comparable to that of the phenyl ring of Phe82 in the wild-type protein. Despite this perturbation in structure, solvent accessibility computations show that heme solvent exposure has not increased in the Gly82 variant protein. However, refolding does result in the introduction of a number of polar groups into the hydrophobic heme pocket. This appears to be responsible for the decreased reduction potential of the heme in this protein. The present study, along with that of the Ser82 variant protein (Louie et al., 1988b), clearly establishes the link between dielectric constant within the heme crevice and reduction potential. The further anomalously low electron transfer activity of the Gly82 variant protein would appear to arise from two factors. First, the polypeptide chain medium now adjacent to the heme is unable to facilitate electron transfer in a manner similar to that of the aromatic side-chain of Phe82. Second, polypeptide chain refolding significantly alters the surface contour of the Gly82 protein rendering it less suitable to interact with the corresponding complementary surfaces of redox partners. Our data support the conclusion that Phe82 plays a number of roles in the electron transfer process mediated by yeast iso-1-cytochrome c. These include the maintenance of the heme environment, provision of an optimal medium along the path of electron transfer and formation of interactions at the contact interface in complexes with redox partners.     

Cytochrome c at charged interfaces. 2. Complexes with negatively charged macromolecular systems studied by resonance Raman spectroscopy

We have analyzed the structure of cytochrome c (cyt c) bound in a variety of complexes in which negatively charged molecular groups interact with the positively charged binding domain around the heme crevice of cyt c. Using resonance Raman spectroscopy, we could demonstrate that these interactions induce the same conformational changes as they were observed in the surface-enhanced resonance Raman experiments of cyt c adsorbed on the Ag electrode [Hildebrandt & Stockburger (1989) Biochemistry (preceding paper in this issue)]. When cyt c is bound to (As4W40O140)27-, state II is stabilized, whereas in complexes with phosvitin and cytochrome b5 state I is formed. The complexes with phospholipid vesicles and inverted micelles reveal a mixture of both states. It is suggested that these systems as well as cyt c adsorbed on the Ag electrode may be regarded as model systems for the physiological complexes of cyt c with cytochrome oxidase and cytochrome reductase. On the basis of our findings it is proposed that the biological electron-transfer reactions are controlled by electric field induced conformational transitions of cyt c upon complex formation with its physiological redox partners.     

Resonance Raman investigations of cytochrome c conformational change upon interaction with the membranes of intact and Ca2+-exposed mitochondria

The conformational states of cytochrome c inside intact and Ca(2+)-exposed mitochondria have been investigated using resonance Raman spectroscopy. Intact and swelling bovine heart and rat liver mitochondria were examined with an excitation wavelength (413.1 nm) in resonance with the Soret transition of ferrous cytochrome c. The different b- to c-type cytochrome concentration ratio in mitochondria from two different tissues was used to help assign the Raman spectral components. Resonance Raman spectra were also recorded for mitochondria fractions (supernatants and pellets) obtained from swollen (Ca(2+)-exposed) mitochondria after differential centrifugation. The results illustrate that cytochrome c has an altered vibrational spectrum in solution, in intact, and in swollen mitochondria. When cytochrome c is released from mitochondria, its Raman spectrum becomes identical to that of ferrous cytochrome c in solution. The spectra of mitochondrial pellets indicate that a small amount of structurally modified cytochrome c remains associated with the heavy membrane fraction. Indeed, spectroscopic shifts in the low-frequency fingerprint and the high-frequency marker-band regions suggest that membrane binding leads to a partial opening of the heme pocket and an alteration of the heme thioether bonds. The results support the conclusion that most cytochrome c molecules in mitochondria are membrane-bound and that the cytochrome c structure changes upon binding. Furthermore, changes in the resonance Raman active mode located at 675 cm(-)(1) in the spectra of intact, swollen, and fractionated mitochondria indicate that b-type cytochromes may also undergo structural alterations during mitochondrial swelling and disruption.     

Ligand dynamics in an electron transfer protein. Picosecond geminate recombination of carbon monoxide to heme in mutant forms of cytochrome c

Substitution of the heme coordination residue Met-80 of the electron transport protein yeast iso-1-cytochrome c allows external ligands like CO to bind and thus increase the effective redox potential. This mutation, in principle, turns the protein into a quasi-native photoactivable electron donor. We have studied the kinetic and spectral characteristics of geminate recombination of heme and CO in a series of single M80X (X = Ala, Ser, Asp, Arg) mutants, using femtosecond transient absorption spectroscopy. In these proteins, all geminate recombination occurs on the picosecond and early nanosecond time scale, in a multiphasic manner, in which heme relaxation takes place on the same time scale. The extent of geminate recombination varies from >99% (Ala, Ser) to approximately 70% (Arg), the latter value being in principle low enough for electron injection studies. The rates and extent of the CO geminate recombination phases are much higher than in functional ligand-binding proteins like myoglobin, presumably reflecting the rigid and hydrophobic properties of the heme environment, which are optimized for electron transfer. Thus, the dynamics of CO recombination in cytochrome c are a tool for studying the heme pocket, in a similar way as NO in myoglobin. We discuss the differences in the CO kinetics between the mutants in terms of the properties of the heme environment and strategies to enhance the CO escape yield. Experiments on double mutants in which Phe-82 is replaced by Asp or Gly as well as the M80D substitution indicate that such steric changes substantially increase the motional freedom-dissociated CO.     

Resonance Raman study on the structure of the active sites of microsomal cytochrome P-450 isozymes LM2 and LM4

The isozymes 2 and 4 of rabbit microsomal cytochrome P-450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100-1700 cm-1 is presented for different ferric states of cytochrome P-450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme-protein interactions. While in cytochrome P-450 LM2 the six-coordinated low-spin configuration is predominantly occupied, in the isozyme LM4 the five-coordinated high-spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low-spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high-spin form of the isozyme LM4. The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type-I substrate binding. While in cytochrome P-450 LM4 the occupation of the substrate-binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground-state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accommodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high-spin states in the substrate-bound and substrate-free enzyme are structurally different. The formation of the inactive form, P-420, involves a severe structural rearrangement in the heme binding pocket leading to drastic changes of the vinyl group conformations. The conformational differences of the active sites in cytochromes P-450 LM2 and LM4 observed in this work contribute to the understanding of the structural basis accounting for substrate and product specificity of cytochrome P-450 isozymes.     

The cytochrome c oxidase-cytochrome c complex: spectroscopic analysis of conformational changes in the protein-protein interaction domain

Binding to cytochrome c oxidase induces a conformational change in the cytochrome c molecule. This conformational change has been characterized by comparing the binding of native cytochrome c and chemically modified cytochrome c derivatives to bovine cytochrome c oxidase by using absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopy. The following derivatives were analyzed: (i) cytochrome c modified at all 19 lysine residues to yield the (N epsilon-acetimidyl)19 cytochrome c, (N epsilon-isopropyl)19 cytochrome c, and (N epsilon,N epsilon-dimethyl)19 cytochrome c; (ii) cytochrome c in which Met65 and Met80 are converted to the methionine sulfoxide; (iii) cytochrome c with a single break in the polypeptide chain at Arg38 or Gly37. The derivatives bind to cytochrome c oxidase at a ratio of one heme c per heme aa3. The association constants are similar to that of native cytochrome c except for (N epsilon-isopropyl)19 and (N epsilon,N epsilon-dimethyl)19 cytochromes c, which bind respectively four times and six times less strongly. The derivatives are good substrates for the cytochrome c oxidase reaction. The spectral changes accompanying the binding of the modified cytochromes c to cytochrome c oxidase are quite different from the spectral changes observed with native cytochrome c. The different optical absorption and MCD changes are explained by a polarity change around the exposed heme edge in the cytochrome c-cytochrome c oxidase complex. The CD changes indicate a conformational rearrangement restricted to the surface area surrounding the exposed heme edge. The rearrangement may involve a movement of the evolutionarily conserved Phe82 out of the vicinity of the heme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of leucine 85 to the structure and function of Saccharomyces cerevisiae iso-1 cytochrome c.

Cytochrome c is a small electron-transport protein whose major role is to transfer electrons between complex III (cytochrome reductase) and complex IV (cytochrome c oxidase) in the inner mitochondrial membrane of eukaryotes. Cytochrome c is used as a model for the examination of protein folding and structure and for the study of biological electron-transport processes. Amongst 96 cytochrome c sequences, residue 85 is generally conserved as either isoleucine or leucine. Spatially, the side chain is associated closely with that of the invariant residue Phe82, and this interaction may be important for optimal cytochrome c activity. The functional role of residue 85 has been examined using six site-directed mutants of Saccharomyces cerevisiae iso-1 cytochrome c, including, for the first time, kinetic data for electron transfer with the principle physiological partners. Results indicate two likely roles for the residue: first, heme crevice resistance to ligand exchange, sensitive to both the hydrophobicity and volume of the side chain; second, modulation of electron-transport activity through maintenance of the hydrophobic character of the protein in the vicinity of Phe82 and the exposed heme edge, and possibly of the ability of this region to facilitate redox-linked conformational change.     

Picosecond structural dynamics of myoglobin following photodissociation of carbon monoxide as revealed by ultraviolet time-resolved resonance Raman spectroscopy

Picosecond protein dynamics of myoglobin in response to structural changes in heme upon CO dissociation were observed in a site-specific fashion for the first time using time-resolved UV resonance Raman spectroscopy. Transient UV resonance Raman spectra showed several phases of intensity changes in both tryptophan and tyrosine Raman bands. Five picoseconds after dissociation, the W18, W16, and W3 bands of tryptophan residues and the Y8a band of tyrosine residues decreased in intensity, followed by recovery of the Y8a band intensity in hundreds of picoseconds and recovery of the tryptophan bands in nanoseconds. These spectral changes suggest that the change in heme structure impulsively drives concerted movement of the EF helical section and that rearrangements toward a deoxy structure occur in the heme vicinity and in the A helix within a time frame of sub-nanoseconds to nanoseconds.     

Mutation of residues critical for benzohydroxamic acid binding to horseradish peroxidase isoenzyme C.

Aromatic substrate binding to peroxidases is mediated through hydrophobic and hydrogen bonding interactions between residues on the distal side of the heme and the substrate molecule. The effects of perturbing these interactions are investigated by an electronic absorption and resonance Raman study of benzohydroxamic acid (BHA) binding to a series of mutants of horseradish peroxidase isoenzyme C (HRPC). In particular, the Phe179 --> Ala, His42 --> Glu variants and the double mutant His42 --> Glu:Arg38 --> Leu are studied in their ferric state at pH 7 with and without BHA. A comparison of the data with those previously reported for wild-type HRPC and other distal site mutants reaffirms that in the resting state mutation of His42 leads to an increase of 6-coordinate aquo heme forms at the expense of the 5-coordinate heme state, which is the dominant species in wild-type HRPC. The His42Glu:Arg38Leu double mutant displays an enhanced proportion of the pentacoordinate heme state, similar to the single Arg38Leu mutant. The heme spin states are insensitive to mutation of the Phe179 residue. The BHA complexes of all mutants are found to have a greater amount of unbound form compared to the wild-type HRPC complex. It is apparent from the spectral changes induced on complexation with BHA that, although Phe179 provides an important hydrophobic interaction with BHA, the hydrogen bonds formed between His42 and, in particular, Arg38 and BHA assume a more critical role in the binding of BHA to the resting state.     

Intermediate and stable redox states of cytochrome c studied by low temperature resonance Raman spectroscopy

Stabilized intermediate redox states of cytochrome c are generated by radiolytic reduction of initially oxidized enzyme in glass matrices at liquid nitrogen temperature. In the intermediate states the heme group is reduced by hydrated electrons, whereas the protein conformation is restrained close to its oxidized form by the low-temperature glass matrix. The intermediate and stable redox states of cytochrome c at neutral and alkaline pH are studied by low-temperature resonance Raman spectroscopy using excitations in resonance with the B (Soret) and Q1 (beta) optical transitions. The assignments of the cytochrome c resonance Raman bands are discussed. The observed spectral characteristics of the intermediate states as well as of the alkaline transition in the oxidized state are interpreted in terms of oxidation-state marker modes, spin-state marker modes, heme iron--axial ligand stretching modes, totally symmetric in-plane porphyrin modes, nontotally symmetric in-plane modes, and out-of-plane modes.     

Comparison of C40/82A and P27A C40/82A barstar mutants using 19F NMR

Barstar, an inhibitor of the enzyme barnase, contains two phenylalanine residues, three tryptophan residues, and two proline residues. After incorporating either 2-19F-Phe, 4-19F-Phe, or 6-19F-Trp, the structural, dynamic, and folding properties of two mutants (C40/82A, a double mutant, and P27A C40/82A, a triple mutant) were studied by 19F NMR. Experiments were performed as a function of temperature and urea with the two mutants. We show that the consequences of the P27A mutation are extensive. The effect of the mutation is transmitted to distant residues (Phe56 and Trp53) as well as to a residue deeply buried in the hydrophobic core (Phe74). By incorporating 2-19F-Phe, it is shown that Phe56 undergoes a slow ring flipping on the NMR time scale in the triple mutant that is not observed in the double mutant. On the other hand, incorporating 4-19F-Phe shows that the P27A mutation has little effect along the Cbeta-Cgamma axis of Phe56. Labeling with 4-19F-Phe shows, from line broadening, that Phe74 experiences more dynamic motion than does Phe56 in both the double and triple mutant. After incorporating 6-19F-Trp, it is found that, in the triple mutant, Trp53 shows conformational heterogeneity at low temperature while Trp44, which is close to the P27A mutation, does not. At 20 degrees C, residual native-like structure was detected around Trp53 at high concentrations of denaturant. Barstar is cold denatured in the presence of urea. For the double mutant at temperatures below 15 degrees C, and in the presence of 2.5-3.5 M urea, the resonance for Phe74 broadens, and two peaks are observed at 5 degrees C indicative of an exchange process. From line-shape analysis, assuming a two-site conformational exchange, the rate constants as a function of temperature can be extracted. An Eyring plot is linear at 0 M urea but deviates from linearity below 20 degrees C in the presence of 2.5 or 3.5 M urea. The data as a function of urea suggest sequential events in the unfolding process.     

Controlling conformational flexibility of an O₂-binding H-NOX domain

Heme Nitric oxide/OXygen binding (H-NOX) domains have provided a novel scaffold to probe ligand affinity in hemoproteins. Mutation of isoleucine 5, a conserved residue located in the heme-binding pocket of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX), was carried out to examine changes in oxygen (O(2))-binding properties. A series of I5 mutants (I5F, I5F/I75F, I5F/L144F, I5F/I75F/L144F) were investigated to probe the role of steric bulk within the heme pocket. The mutations significantly increased O(2) association rates (1.5-2.5-fold) and dissociation rates (8-190-fold) as compared to wild-type Tt H-NOX. Structural changes that accompanied the I5F mutation were characterized using X-ray crystallography and resonance Raman spectroscopy. A 1.67 ? crystal structure of the I5F mutant indicated that introducing a phenylalanine at position 5 resulted in a significant shift of the N-terminal domain of the protein, causing an opening of the heme pocket. This movement also resulted in an increased amount of flexibility at the N-terminus and the loop covering the N-terminal helix as indicated by the two conformations of the first six N-terminal amino acids, high B-factors in this region of the protein, and partially discontinuous electron density. In addition, introduction of a phenylalanine at position 5 resulted in increased flexibility of the heme within the pocket and weakened hydrogen bonding to the bound O(2) as measured by resonance Raman spectroscopy. This study provides insight into the critical role of I5 in controlling conformational flexibility and ligand affinity in H-NOX proteins.     

Polyanion binding to cytochrome c probed by resonance Raman spectroscopy

The interaction of ferricytochrome c with negatively charged heteropolytungstates was studied by resonance Raman spectroscopy. In analogy to previous findings on ferricytochrome c bound to other types of charged interface (Hildebrandt, P. and Stockburger, M. (1989) Biochemistry 28, 6710-6721, 6722-6728), it was shown that in these complexes the conformational states I and II are stabilized. While in state I, the structure is the same as is in the uncomplexed heme protein, in state II three different coordination configurations coexist, i.e., a six-coordinated low-spin, a five-coordinated high-spin and a six-coordinated high-spin form. These configurations constitute thermal coordination equilibria whose thermodynamic properties were determined. The detailed analysis of the low-frequency resonance Raman spectra reveals that in state II the heme pocket assumes an open structure leading to a significantly higher flexibility of the heme group compared to the native ferricytochrome c. It is concluded that these structural changes are the result of Coulombic attractions between the polyanions and the lysine residues around the exposed heme edge which destabilize the heme crevice. Modifications of these interactions upon variation of the ionic strength, the pH or the type of the polytungstate are sensitively reflected by changes of the coordination equilibria in state II as well as of the conformational equilibrium of state I and state II. The conformational changes in state II significantly differ from those associated with the alkaline transition of ferricytochrome c. However, there are some structural similarities between the acid form of the heme protein stable below pH 2.5 in aqueous solution and the six-coordinated high-spin configuration of the bound ferricytochrome c at neutral pH (state II). This suggests that electrostatic interactions with the heteropolytungstates perturb the ionic equilibria of those amino acid side chains which are involved in the acid-induced transition leading to a significant upshift of the apparent pKa.     

X-ray crystallography, CD and kinetic studies revealed the essence of the abnormal behaviors of the cytochrome b5 Phe35-->Tyr mutant.

Conserved phenylalanine 35 is one of the hydrophobic patch residues on the surface of cytochrome b5 (cyt b5). This patch is partially exposed on the surface of cyt b5 while its buried face is in direct van der Waals' contact with heme b. Residues Phe35 and Phe/Tyr74 also form an aromatic channel with His39, which is one of the axial ligands of heme b. By site-directed mutagenesis we have produced three mutants of cyt b5: Phe35-->Tyr, Phe35-->Leu, and Phe35-->His. We found that of these three mutants, the Phe35-->Tyr mutant displays abnormal properties. The redox potential of the Phe35-->Tyr mutant is 66 mV more negative than that of the wild-type cyt b5 and the oxidized Phe35-->Tyr mutant is more stable towards thermal and chemical denaturation than wild-type cyt b5. In this study we studied the most interesting mutant, Phe35-->Tyr, by X-ray crystallography, thermal denaturation, CD and kinetic studies of heme dissociation to explore the origin of its unusual behaviors. Analysis of crystal structure of the Phe35-->Tyr mutant shows that the overall structure of the mutant is basically the same as that of the wild-type protein. However, the introduction of a hydroxyl group in the heme pocket, and the increased van der Waals' and electrostatic interactions between the side chain of Tyr35 and the heme probably result in enhancement of stability of the Phe35-->Tyr mutant. The kinetic difference of the heme trapped by the heme pocket also supports this conclusion. The detailed conformational changes of the proteins in response to heat have been studied by CD for the first time, revealing the existence of the folding intermediate.     

Reactivity of manganese peroxidase: site-directed mutagenesis of residues in proximity to the porphyrin ring

The purpose of this study was to determine the effect of heme pocket hydrophobicity on the reactivity of manganese peroxidase. Residues within 5 A of the heme active site were identified. From this group, Leu169 and Ser172 were selected and mutated to Phe and Ala, respectively. The mutant proteins were then characterized by steady-state kinetics. Whereas the Leu169Phe mutation had little, if any, effect on activity, the Ser172Ala mutation decreased kcat and also the specificity constant (kcat/Km) for Mn2+, but not H2O2. Transient-state studies indicated that the mutation affected only the reactions of compound II. These results indicate that compound II is the most sensitive to changes in the heme environment.     

Raman spectra of heme a, cytochrome oxidase-ligand complexes, and alkaline denatured oxidase.

We report 441.6 nm excitation resonance Raman spectra of oxidized and reduced monomeric heme a-imidazole, cytochrome oxidase-exogenous ligand complexes in various redox states, and alkaline denatured oxidase. These data show that, in reduced oxidase, the cytochrome a3 Raman spectrum has bands at 215, 364, 1230, and 1670 cm-1 not observed in the cytochrome a spectrum. The appearance of these bands in the reduced cytochrome a3 spectrum is due to interactions between the heme a of cytochrome a3 and its protein environment and not to intrinsic properties of heme a. These interactions are pH sensitive and strongly influence the vibrational spectra of both heme a groups. We assign the 1670-cm-1 band to the heme a formyl substituent and propose that the intensity of the 1670 cm-1 is high for reduced cytochrome a3 because the C==O lies in the porphyrin plane and is very weak for oxidized and reduced cytochrome a, oxidized cytochrome a3, and oxidized and reduced heme a-imidazole because the C==O lies out of the plane. We suggest that movement of the C==O in and out of the plane explains the ligand induced spectral shift in the optical absorption spectrum of reduced cytochrome a3. Finally, we confirm the observation of Adar & Yonetani (private communication) that, under laser illumination, resting oxidase is photoreactive.     

Structural rationalization of novel drug metabolizing mutants of cytochrome P450 BM3

Three newly discovered drug metabolizing mutants of cytochrome P450 BM3 (van Vugt-Lussenburg et al., Identification of critical residues in novel drug metabolizing mutants of Cytochrome P450 BM3 using random mutagenesis, J Med Chem 2007;50:455-461) have been studied at an atomistic level to provide structural explanations for a number of their characteristics. In this study, computational methods are combined with experimental techniques. Molecular dynamics simulations, resonance Raman and UV-VIS spectroscopy, as well as coupling efficiency and substrate-binding experiments, have been performed. The computational findings, supported by the experimental results, enable structural rationalizations of the mutants. The substrates used in this study are known to be metabolized by human cytochrome P450 2D6. Interestingly, the major metabolites formed by the P450 BM3 mutants differ from those formed by human cytochrome P450 2D6. The computational findings, supported by resonance Raman data, suggest a conformational change of one of the heme propionate groups. The modeling results furthermore suggest that this conformational change allows for an interaction between the negatively charged carboxylate of the heme substituent and the positively charged nitrogen of the substrates. This allows for an orientation of the substrates favorable for formation of the major metabolite by P450 BM3.