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

Hildebrandt, Peter; Stockburger, Manfred

doi:10.1021/bi00442a027

Cited by 92 publications

(59 citation statements)

References 25 publications

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“…In AOT reverse micelles, strong electrostatic interactions occur between the anionic polar head groups and the positively charged protein residues (pI=10.5). The spectroscopic results previously reported by 29 have been interpreted as originating from the opening of the CYTC heme crevice under the electrostatic field generated by the micellar anionic head groups, since most of the CYTC positive charges are located around the heme crevice [25][26][27] . Our simulations support this picture.…”

Section: Discussionmentioning

confidence: 99%

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Molecular dynamics simulations of cytochrome c unfolding in AOT reverse micelles: The first steps

2010

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2This paper explores the reduced form of horse cytochrome c confined in reverse micelles (RM) of sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) in isooctane by molecular dynamics simulation. RMs of two sizes were constructed at a water content of W o = [H 2 O]/[AOT] = 5.5 and 9.1. Our results show that the protein secondary structure and the heme conformation both depend on micellar hydration. At low hydration, the protein structure and the heme moiety remain stable, whereas at high water content the protein becomes unstable and starts to unfold. At W o = 9.1, according to the X-ray structure, conformational changes are mainly localized on protein loops and around the heme moiety, where we observe a partial opening of the heme crevice. These findings suggest that within our time window (10 ns), the structural changes observed at the heme level are the first steps of the protein denaturation process, previously described experimentally in micellar solutions. In addition, a specific binding of AOT molecules to a few lysine residues of the protein was found only in the small-sized RM.3

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Section: Discussionmentioning

confidence: 99%

“…Depending on the microenvironment, CYTC can adopt a number of conformational states. For example, membranebound CYTC has been thoroughly investigated [25][26][27] . It was shown that the membrane anionic environ-ment disrupts the native protein structure, leading to molten globule-like states 26,28 .…”

Section: Introductionmentioning

confidence: 99%

Molecular dynamics simulations of cytochrome c unfolding in AOT reverse micelles: The first steps

2010

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“…In order to detect subtle spectral differences between the various samples, a Raman difference technique was employed which is described in detail elsewhcre [21,221. The protein was dcposited in a rotating cell in order to minimize thermal heating by the exciting laser beam (20mW at the sample).…”

Section: Methodsmentioning

confidence: 99%

“…The signal/noise ratio was improved by repetilivc scanning giving a total accumulation time of about 30 s per monochrometer setting. The spectra were analyzed by a band-fitting program which is described in [21]. HzOzdependent benzphetamine N-demethylation and aniline p-hydroxylation catalyzed by LM2,,,, and LM2,1i, were determined by measuring formaldehyde and p-aminophenol formation, respectively.…”

Section: Methodsmentioning

confidence: 99%

Protein–protein interactions in microsomal cytochrome P‐450 isozyme LM2 and their effect on substrate binding

Hildebrandt

Garda

Stier

et al. 1989

European Journal of Biochemistry

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The effects of protein ~ protein interactions and substrate binding on the structure of the active site of rabbit liver microsomal cytochrome P-450 LM2 have been analyzed by resonance Kaman spectroscopy of the monomeric and oligomeric protein in solution. Also Hz02-dependent catalytic activities of the two states have been compared.The two vinyl substituents of the heme exhibit different orientations, as indicated by the frequencies and intensities of their stretching vibrations. One group lies in the plane of the heme and remains unchanged in the two states of cytochrome P-450 LM2, the other is tilted out of the plane. The tilting angle in oligomers was smaller than in monomers. These vinyl stretching modes together with some porphyrin modes, were found to be sensitive indicators of the quaternary structure and of substrate binding. In both the oligomer and the monomer, substrate binding causes changes of the relative intensities of some porphyrin modes and the vinyl stretching vibrations which may reflect modifications of the electronic transitions due to hydrophobic interactions between the bound substrate and the heme. In contrast to the monomeric cytochrome P-450 LM2, benzphetamine binding to the oligomcrs of this isozyme additionally produces a shift of the spin-state equilibrium. This indicates that in the oligomer the substrate-binding pocket is converted by protein -protein interaction to a structure that forces substrates to interfere with the sixth ligands, inducing an increase of the five-coordinated high-spin configuration. In the monomcr the substrate-binding pocket can accommodate benzphetamine without affecting the spin state. Binding of imidazole to the monomeric and oligomeric cytochrome P-450 LM2 produces essentially the same resonance Kaman spectra. Apparently the replacement of the native sixth ligand by imidazole disturbs the structure of the active site in such a way that it becomes insensitive to protein-protein interactions.Hz02-dependent IV-demethylation of bcnzphetamine and aniline p-hydroxylation by cytochrome P-450 LM2 did not depend on its state of aggregation.In mammalian liver miocrosomes a large variety of xenobiotics are oxygenated by an enzyme complex whose central protein is cytochrome P-450 [I]. This heme protein runs through a cyclic process including substrate binding. electron transfer. and oxygen activation. So far no details are known about the molecular mechanism of individual reaction steps. Recently, howcver, good progress has been made in the elucidation of the structure/function relationship of bacterial cytochrome P-450 ( P-450,,,n) whose crystal structure has been determined to 0.16-nm resohition [2]. Thus important information could be obtained about the conformation of the active site and the substrate-binding pocket and the modes of interaction of the substrate with the heme. Based on sequence alignments of eukaryotic cytochrome P-450 and P-450,,,, it was concluded that essential structural features should be largely conserved [2 -41. A fundamental difference o...

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“…It has been proposed hitherto that the conformational change in each oxidation state of cyt c provides the driving force for the dissociation process. [53][54][55] It is difficult to detect structural differences in examples of flat and well-ordered monolayers by using promoter molecules. [16][17][18][19][20][21][22] In fact, in the case of 4,4'-bipyridine, the kinetic parameters of the reduction and oxidation reactions of cyt c, which were estimated by using rotating-disk and ring-disk electrodes, showed no difference.…”

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confidence: 99%

Electron‐Transfer Reactions through the Associated Interaction between Cytochrome c and Self‐Assembled Monolayers of Optically Active Cobalt(III) Complexes: Molecular Recognition Ability Induced by the Chirality of the Cobalt(III) Units

Takahashi¹,

Inomata²,

Funahashi³

et al. 2007

Chemistry A European J

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Self-assembled monolayers (SAMs) of optically active Co(III) complexes ((S)-2/(R)-2) that contain (S)- or (R)-phenylalanine derivatives as a molecular recognition site were constructed on Au electrodes ((S)-2-Au/(R)-2-Au). Molecular recognition characteristics induced by the S and R configurations were investigated by measurements of electron-transfer reactions with horse heart cytochrome c (cyt c). The electrochemical studies indicate that the maximum current of cyt c reduction is obtained when the Au electrode is modified by 2 with a moderate coverage of approximately 4.0 x 10(-11) mol cm(-2). Since the Au electrode is not densely packed with the Co(III) units at this concentration, we conclude that the penetrative association process between cyt c and the Co(III) unit plays an important role in this electron-transfer system. The differences in the electron-transfer rates of (S)-2-Au and (R)-2-Au increase with increasing scan rates, a result indicating that the chiral ligand has an influence on the rate of association of the complexes with cyt c. 3-Au has a mixed monolayer composed of 2 and hexanethiol and exhibits electron-transfer behavior comparable to 2-Au. The difference in the association rates of (S)-3-Au and (R)-3-Au is larger than that between (S)-2-Au and (R)-2-Au, which indicates that the molecular recognition ability of 3-Au has been enhanced by filling the gap between molecules of 2 with hexanethiols. The differences in the oxidation rates of cyt c(II) between (S)-2-Au and (R)-2-Au and between (S)-3-Au and (R)-3-Au were larger than the differences in the rates of the reduction of cyt c(III); this suggests that the size of the heme crevice varies according to the oxidation state of cyt c.

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

Cited by 92 publications

References 25 publications

Molecular dynamics simulations of cytochrome c unfolding in AOT reverse micelles: The first steps

Molecular dynamics simulations of cytochrome c unfolding in AOT reverse micelles: The first steps

Protein–protein interactions in microsomal cytochrome P‐450 isozyme LM2 and their effect on substrate binding

Electron‐Transfer Reactions through the Associated Interaction between Cytochrome c and Self‐Assembled Monolayers of Optically Active Cobalt(III) Complexes: Molecular Recognition Ability Induced by the Chirality of the Cobalt(III) Units

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