Christiane Jung

Insight into protein structure and protein-ligand recognition by Fourier transform infrared spectroscopy.

An overview of the application of Fourier transform infrared spectroscopy for the analysis of the structure of proteins and protein–ligand recognition is given. The principle of the technique and of the spectra analysis is demonstrated. Spectral signal assignments to vibrational modes of the peptide chromophore, amino acid side chains, cofactors and metal ligands are summarized. Several examples for protein–ligand recognition are discussed. A particular focus is heme proteins and, as an example, studies of cytochrome P450 are reviewed. Fourier transform infrared spectroscopy in combination with the various techniques such as time‐resolved and low‐temperature methods, site‐directed mutagenesis and isotope labeling is a helpful approach to studying protein–ligand recognition. Copyright

The mystery of cytochrome P450 Compound I: A mini-review dedicated to Klaus Ruckpaul

The cytochrome P450 protein-bound porphyrin complex with the iron-coordinated active oxygen atom is called Compound I, which is presumably the intermediate species which hydroxylates inert carbon-hydrogen bonds of substrates. In this mini-review, the milestones in discovering Compound I of cytochrome P450 are summarized. It will be discussed what was known and suggested in the years before 1984, the year when Klaus Ruckpauls first book about cytochrome P450 appeared, and compared with recent approaches and studies to catch and characterize this intermediate oxygen species in the reaction cycle of cytochrome P450. Although many studies have been undertaken before and after 1984 to characterize Compound I, its electronic structure and physicochemical properties are still a mystery. The conclusion from this review is that the knowledge about Compound I has significantly increased; however, we still ask the same questions. There is a need for improved experimental approaches, detection techniques, and theoretical simulations for future studies of cytochrome P450 Compound I. This mini-review is dedicated to Klaus Ruckpaul on the occasion of his 80th birthday.

Intermediates in the reaction of substrate-free cytochrome P450cam with peroxy acetic acid

Freeze‐quenched intermediates of substrate‐free cytochrome 57Fe‐P450cam in reaction with peroxy acetic acid as oxidizing agent have been characterized by EPR and Mössbauer spectroscopy. After 8 ms of reaction time the reaction mixture consists of ∼90% of ferric low‐spin iron with g‐factors and hyperfine parameters of the starting material; the remaining ∼10% are identified as a free radical (S′=1/2) by its EPR and as an iron(IV) (S=1) species by its Mössbauer signature. After 5 min of reaction time the intermediates have disappeared and the Mössbauer and EPR‐spectra exhibit 100% of the starting material. We note that the spin‐Hamiltonian analysis of the spectra of the 8 ms reactant clearly reveals that the two paramagnetic species, e.g. the ferryl (iron(IV)) species and the radical, are not exchanged coupled. This led to the conclusion that under the conditions used, peroxy acetic acid oxidized a tyrosine residue (probably Tyr‐96) into a tyrosine radical (Tyr‐96), and the iron(III) center of substrate‐free P450cam to iron(IV).

Crystal structure of cytochrome P-450cam complexed with the (1S)-camphor enantiomer.

The crystal structure of cytochrome P‐450cam complexed with the enantiomer (1S)‐camphor has been solved to 1.8 Å resolution and compared with the structure of the (1R)‐camphor P‐450cam complex. The overall protein structure is the same for both enantiomer complexes. However, the orientation of the substrates in the heme pocket differs. In contrast to (1R)‐camphor, the (1S)‐enantiomer binds in at least two orientations. The major binding mode of (1S)‐camphor resembles the one of the (1R)‐enantiomer in that there is a hydrogen bond between Tyr‐96 and the quinone group of camphor, and the 10‐methyl group points towards the I‐helix. The binding differs in that C‐5 is not at a position suitable for hydroxylation. In the other orientation (1S)‐camphor is not hydrogen bonded, but C‐5 is located suitably for hydroxylation.

The high-spin/low-spin equilibrium in cytochrome P-450 — A new method for determination of the high-spin content

A new method for determination of the population of the high-spin state (high-spin content) in ferric cytochrome P-450 is presented. Based on curve fitting the electronic absorption spectra with a linear combination of gaussian bands analytical functions for the pure high-spin and pure low-spin states were constructed. These functions were used to fit the high-spin/low-spin mixed spectra. A good fit of the absorption spectra of six different cytochrome P-450 proteins in the presence and absence of substrates was found, indicating a similar pi-electron structure of the porphyrin and a similar chemical nature of the nearest coordination sphere of the iron in all cytochrome P-450 proteins.

Spectroscopic characterization of the iron-oxo intermediate in cytochrome P450

Abstract From analogy to chloroperoxidase from Caldariomyces fumago, it is believed that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of cytochrome P450 corresponds to an iron(IV) porphyrin-π-cation radical (compound I). However, our recent studies on P450cam revealed that after 8 ms a tyrosine radical and iron(IV) were formed in the reaction of ferric P450 with external oxidants in the shunt pathway. The present study on the heme domain of P450BM3 (P450BMP) shows a similar result. In addition to a tyrosine radical, a contribution from a tryptophan radical was found in the electron paramagnetic resonance (EPR) spectra of P450BMP. Here we present comparative multi-frequency EPR (9.6, 94 and 285 GHz) and Mössbauer spectroscopic studies on freeze-quenched intermediates produced using peroxy acetic acid as oxidant for both P450 cytochromes. After 8 ms in both systems, amino acid radicals occurred instead of the proposed iron(IV) porphyrin-π-cation radical, which may be transiently formed on a much faster time scale. These findings are discussed with respect to other heme thiolate proteins. Our studies demonstrate that intramolecular electron transfer from aromatic amino acids is a common feature in these enzymes. The electron transfer quenches the presumably transiently formed porphyrin-π-cation radical, which makes it extremely difficult to trap compound I.

Mobility of norbornane-type substrates and water accessibility in cytochrome P-450cam

The behaviour of norbornane-type substrates bound to oxidised cytochrome P-450cam (CYP 101) in 60% (w/w) glycerol-containing phosphate buffer was investigated using electronic absorption spectroscopy. The high-pressure dependence study revealed that the value of the spin-state reaction-volume change decreased from -70 to -22.8 cm3/mol with decreasing high-spin state content from 99 to 63%. Simultaneously, the values for the enthalpy and entropy determined from the low-temperature dependence of the spin-state transition decreased from 73.7 to 24.3 kJ/mol and from 310.4 to 88.9 J/mol K, respectively. Under our experimental conditions the pH-value of the buffer remained at low temperatures and high pressures in the range of pH 7-8, in which no pH-value-induced spin-state conversion occurred. Therefore, the secondary effect of the temperature and pressure-induced pH change can be disregarded as being responsible for the observed spin-state transition effects. Substrate dissociation constants were determined. From the temperature-jump experiments (297 K to 180 K) we found a higher mobility in the active site for the substrates in the sequence (1R)-camphor, (1S)-camphor, camphane, (1R)- and (1S)-camphorquinone, norcamphor, and norbornane. Our findings can be explained by the incomplete fit of the methyl groups of the norbornane-type substrate to the protein, in particular to the I-helix, predominantly determining the substrate mobility and water accessibility to the protein.

Cytochrome P-450-CO and substrates: lessons from ligand binding under high pressure

An overview of the application of high-pressure studies on the carbon monoxide complex of cytochrome P-450 is given. Different approaches to characterize ligand binding steps, the conformational states and substates and the compressibility of the ligand-bound complex are reviewed. A particular focus is the effect of substrates on these properties. It is shown that substrate mobility, compressibility and water accessibility are interrelated and may have functional meaning.

Proton nuclear magnetic resonance study of the binary complex of cytochrome P450cam and putidaredoxin: interaction and electron transfer rate analysis.

A 1H nuclear magnetic resonance study of the complex of cytochrome P450cam‐putidaredoxin has been performed. Isocyanide is bound to cytochrome P450cam in order to increase the stability of the protein both in the reduced and the oxidized state. Diprotein complex formation was detected through variation of the heme methyl proton resonances which have been assigned in the two redox states. The electron transfer rate at equilibrium was determinated by magnetization transfer experiments. The observed rate of oxidation of reduced cytochrome P450 by the oxidized putidaredoxin is 27 (±7) per s.

p-Nitrophenetole deethylase activity of rat liver microsomes entrapped in polyelectrolyte capsules.

Liver microsomes from phenobarbital induced rats are entrapped in capsules prepared from polyelectrolytes. A comparative analysis of the deethylase activity against p-nitrophenetole by encapsulated and freely suspended microsomes is carried out. The pH optimum occurs at about 7.2 for encapsulated as well as free microsomes. The pH activity profile of encapsulated microsomes, however, is strongly flattened. The maximal velocity of entrapped microsomes is about a quarter of that of free microsomes. The Michaelis-Menten constants are virtually equal. Despite of the lowered activity of encapsulated microsomes this kind of immobilization of enzymes may be useful in biotechnology and medicine because mild immobilization conditions like room temperatures and aqueous solutions are realized.