Semisynthetic flavocytochromes based on cytochrome P450 2B4: reductase and oxygenase activities.

A synthetic flavocytochrome with the reductase and oxygenase activities was obtained by covalent binding of riboflavin to cytochrome P450 2B4. The reactions catalyzed by the newly synthesized flavocytochromes were studied. Formation of carbon monoxide complex with the reduced form of hemoprotein led to 60-80% inhibition of oxygenase reactions, indicating the leading role of reduced heme iron in generating active oxygen species by flavocytochromes.

Design of an artificial hemoprotein based on human serum albumin.

For modelling cytochrome P450-catalyzed reactions, an artificial hemoprotein was designed. Upon complex formation of human serum albumin with iron protoporphyrine IX, there occurred the incorporation of heme into the protein and formation of a specific complex with the albumin to heme molar ratio 2:1. The apparent dissociation constant Kd of the complex, as determined by optical absorption spectroscopic technique, was 1.

Comparative analysis of the secondary structural motifs of P450BM-3 and the regions located upstream of the calmodulin-binding domain in the nitric oxide synthases.

The proposed method of multiple alignment of secondary structures makes it possible to estimate the multidomain enzymes' structural similarity in those cases where, following alignment, the identity appears to be inadequate or too insignificant to estimate protein relationship in spite of their functional analogy. Multiple alignment of sequences, representing the secondary structural elements of the nitric oxide synthase (NOS) N-terminal "tails", localized upstream of the calmodulin (CAM)-binding domain, and the P450 superfamily representative P450BM-3 has revealed the existence of a common secondary structural motif, standing of 60% identity between the polypeptide chain packing of NOS and the full sequence of P450BM3. This fact may point to the existence of their common ancestor.

Scanning tunneling microscopy study of cytochrome P450 2B4 incorporated in proteoliposomes.

In the present paper, the application of scanning tunneling microscopy in cytochrome P450s membrane topology is discussed. The method enables visualization of heme location in the lipid-bilayer-incorporated protein. It is supposed that the membrane-bound cytochrome P450 on the tunneling microscope substrate should behave as 'molecular diode'.

The comparative study of peroxidase activity and substrate binding properties of cytochrome P450 2B4 incorporated into phospholipid vesicles with the use of two different methods.

The comparative study of peroxidase activities and substrate binding properties of cytochrome p450 2B4 in reconstituted vesicles prepared with the use of two different techniques, and microsomal cytochrome P450 was carried out. The data obtained show that the two types of cytochrome P450 2B4-containing proteoliposomes do not differ substantially from each other with respect to H2O2- or cumene hydroperoxide-dependent substrate hydroxylation activities as well as substrate binding properties of hemoprotein reconstituted. However, some parameters measured with proteoliposomal cytochrome P450 are markedly different from those measured with microsomal hemoprotein, suggesting the existence of conformational differences between the molecules of these two cytochromes or the differences in the depth of their immersion into lipid bilayer.

Interaction of organophosphorus analogues of amino acids with P450.

1. This study deals with the oxidation of organophosphorus amino acid analogues by phenobarbital-induced rabbit liver microsomes. It has been shown that 1-aminoalkylphosphonous and 1-aminoalkylthiophosphonic acids are converted by P450 to 1-aminoalkylphosphonic acids.

The identification of the pterin-binding domain in the nitric oxide synthase's sequence.

Comparative studies of primary sequences of nitric oxide synthase (NOS) and pterin-dependent enzymes has shown that the biopterin-dependent site of NOS is localized between the regions responsible for calmodulin and FMN binding. On the example of NOS from rat brain it was demonstrated that this biopterin-binding site is located in the region 750-769. Pairwise alignment of amino acids sequences of NOS from rat brain and rat xantine dehydrogenase revealed that the two proteins are 39.

Membrane topology of N-terminal residues of cytochromes P-450 2B4 and 1A2.

The paper describes the reactivity of fluorescein isothiocyanate towards the N-terminus of cytochromes P450 2B4 and 1A2 in solution, in the natural membrane of microsomes and in proteoliposomes (cholate and ultrasonic). The results obtained indicate, that the N-terminus of microsomal or proteoliposomal cytochromes P-450 2B4 and 1A2 spans the membrane only once and faces the vesicles interior. It was suggested that of major importance in orientation of N-terminal residues in the membrane is not the hydrophobic segment itself but rather the positively charged fragment, following it.

Determination of membrane-bound fragments of cytochrome P-450 2B4.

Membrane-bound sites of cytochrome P-450 2B4 (LM2) were determined by means of two different methods, photoactivated binding of membrane phospholipids to the protein and epitope mapping by antibodies. Phospholipids bearing photoreactive labels at different distances from the their polar 'head' were used in the former case. Phosphatidylcholine labelled at the apolar end of the fatty acid chain bound only to the N-terminal region of the hemoprotein.

Semi-artificial hydroxylating enzymes created by flavins binding to cytochrome P450 2B4 and by bleomycin binding to NADPH-cytochrome P450 reductase.

To create a semi-artificial monomolecular oxygenase system, FAD or FMN were covalently bound to cytochrome P450 2B4 as electron donor centers and bleomycin to NADPH-cytochrome P450 reductase as a generator of active oxygen species. The most catalytically active was the conjugate of cytochrome P450 with FMN, able to initiate the reactions of dimethylaniline and aminopyrine demethylation along with the reaction of aniline p-hydroxylation. The conjugate of cytochrome P450 with FAD oxidized these substrates at a much slower rate.

Effects of amino-terminus truncation in human cytochrome P450IID6 on its insertion into the endoplasmic reticulum membrane of Saccharomyces cerevisiae.

A truncated form of cytochrome P450IID6 deprived of 22 NH2-terminal amino acids residues (P450IID6 delta 1-22) was found in both the cytosol and the microsomal fraction of the yeast, Saccharomyces cerevisiae. A reduced CO difference spectrum of this form was characterized by the absence of absorption at 448 nm and weak absorption at 420 nm. Another peculiarity of P450IID6 delta 1-22 expression was its reduced content in the yeast cells compared to that of P450IID6, with the intracellular levels of the corresponding mRNAs being the same.

Determination of the secondary structure of epoxide hydrolase by Raman spectroscopy.

The secondary structure of microsomal epoxide hydrolase was determined by Raman spectroscopy and the effect of the membrane microenvironment studied. The ratios of the four secondary structure contents, alpha-helix: beta-strand:turn:undefined, were found to be 47:24:17:11 and 58:17:15:10 for the solubilized and the membrane-bound epoxide hydrolase, respectively. Based on the spectral analysis in the 2800-2900 cm-1 range, it was concluded that the protein studied produces the disordering effect on the lipid dimyristoylphosphatidylcholine bilayer at 16 degrees C.

Heme maintains catalytically active structure of cytochrome P-450.

Treatment of purified cytochrome P-450 LM2 and its liposome-bound form with hydrogen peroxide led to complete destruction of the P-450 heme. The apoenzyme thus produced could be reconstituted to catalytically active cytochrome P-450 by incubation with hemin, the reconstitution efficiency being 50% for the soluble enzyme and 80% for the liposome-bound enzyme. The removal of heme from the soluble hemoprotein resulted in a 3-fold decrease in the efficiency of its incorporation into sonicated liposomes.

Compensational dependence of activation parameters of membrane enzymes.

The activation parameters of reactions catalyzed by various membrane enzymes have been analyzed. In all cases there was compensational dependence between the enthalpy and the entropy of the reactions catalyzed. The differences found between lipid-activated, lipid-nonactivated, and water-soluble enzymes are discussed.

Secondary structure and membrane topology of cytochrome P450s.

The secondary structure prediction of 19 microsomal cytochrome P450s from two different families was made on the basis of their amino acid sequences. It was shown that there is structural similarity between the heme-binding sites in these enzymes and those in the bacterial P450cam. An average predicted secondary structure of cytochrome P450 proteins with 70% accuracy contains about 46% alpha-helices, 12% beta-sheets, 9% beta-turns, and 33% random coils.

Effect of the microenvironment on the tertiary structure of cytochrome P-450 LM2.

The relation between microenvironment and the tertiary structure of cytochrome P-450 LM2 has been investigated. No complete relaxation to the most active state of the native enzyme took place in the case of membrane-incorporated hemoprotein with three or four intramolecular cross-links. The spatial organization of the enzyme was predicted to determine the cross-link location on the hemoprotein surface and membrane-incorporated parts of the polypeptide chain.