Julian A. Peterson

NADPH-cytochrome P450 reductase

Abstract The rate of reduction of cytochrome P450 in hepatic microsomes in the presence of NADPH has been measured with a dual wavelength stopped-flow spectrophotometer. The results obtained, with microsomes prepared from phenobarbital-pretreated rats, indicate that the reduction process is biphasic and most probably composed of two concurrent first-order reactions. The rate constant for the reduction of cytochrome P450 in the fast phase in the presence of ethylmorphine is 1.74 s−1. Since approximately 50% or more of the cytochrome P450 is reduced in the fast phase under these conditions, the rate of reduction of cytochrome P450 is approximately 150 nmol min−1 (mg of protein)−1. Under similar conditions the rate of ethylmorphine N-demethylation is 8.6 nmol min−1 (mg of protein)−1. Thus the rate-limiting step in ethylmorphine N-demethylation cannot be the introduction of the first electron into cytochrome P450 by NADPH-cytochrome P450 reductase.

Possible higher valence states of cytochrome P-450 during oxidative reactions

Abstract The addition of the organic hydroperoxide, cumene hydroperoxide, to liver microsomes results in the appearance of a transient spectral change associated with cytochrome P-450. In addition, unique electron paramagnetic resonance signals are observed with liver microsomal cytochrome P-450 comparable to signals obtained when peroxides interact with metmyoglobin. It is suggested that higher valence states of cytochrome P-450 may function during the activation of oxygen for the hydroxylation of a variety of xenobiotics.

Cytochrome P-450 Reductase and Cytochrome b5 in Cytochrome P-450 Catalysis

As the readers of this monograph should be aware, the monooxygenase reaction catalyzed by cytochrome P-450 requires the input of two electrons1,2: (1) In mammalian systems these two electrons are derived from NADPH; in the soluble system isolated from the bacterium Pseudomonas putida, NADH is used as the external source of electrons. A schematic representation of the two different types of electron transfer chains which deliver electrons from the reduced pyridine nucleotides to P-450 is shown in Fig. 1. The system, here referred to as Type I, is found embedded in the membranous endoplasmic reticulum of most eukaryotic cell types, while the second general class, Type II, is found in mitochondria and bacteria. The most completely described P-450 system, which is not membrane-bound, is associated with camphor metabolism in P. putida.3 The Type I electron transport chain is composed of a complex flavoprotein which has both FAD and FMN as prosthetic groups.4 FAD serves as the initial electron acceptor from NADPH, while the FMN serves to reduce the P-450.5 Later in this review, we will discuss the involvement of cytochrome b 5 as a possible component of this electron transport chain. In Type II systems, the reduced pyridine nucleotide first reduces an FAD-containing reductase which subsequently transfers electrons one at a time to a 2Fe,2S iron—sulfur protein.

Nitric oxide complexes of cytochrome P-450

The use of nitric oxide as a spin label to probe the electronic structure of the heine prosthetic group in both native and structurally perturbed hemeproteins has received much attention recently. For example, it has been demonstrated that hemoglobin and myoglobin form ferrous NO complexes which exhibit EPR spectra having superhyperfine lines in the gz signal, consistent with electron spin-nuclear spin interaction of the unpaired electron of the bound NO with a trans axial ligand, presumably due to one of the 14N atoms of an imidazole moiety [ 1-4] . Perturbation of the protein structure either by a denaturing agent [1,5] or an allosteric effector [6] appears to result in either distortion or complete breakage of the bond between the heine iron and the trans ligand resulting in a pentacoordinate heine-NO species which has a sharp 3-line gz EPR signal. Other hemeproteins, including horseradish peroxidase (HRP) and cytochrome c peroxidase (CCP), form stable ferric, as well as ferrous, NO complexes [7]. Their ferric-NO complexes appear diamagnetic and therefore have no EPR signals, while their ferrous-NO complexes have EPR spectra indicative of a rhombically-distorted field for the heine-bound NO. The distinctive superhyperfine lines in the gz signal are even more readily apparent than in hemoglobin and myoglobin ferrous-NO complexes. Several brief reports have suggested that microsomal and submicrosomal cytochrome P-450 form both ferricand ferrous-NO complexes [8-12] . However, the observed EPR spectra of the ferricand ferrous-NO complexes were identical [9,10], as well as being very similar to the denatured ferrous-NO complex of hemoglobin. In this preliminary communication we report that the ferrous microsomal cytocbrome P-450-NO complex is unstable and is converted to a species which has an EPR spectrum similar to that of ferrous P-420--NO. We would also like to report some of the electronic and structural properties of the ferricand ferrous-NO complexes of microsomal and bacterial cytochrome P-450, and to explain the differences between these properties and those previously reported in the literature.

Studies of the unique nature of the cytochrome P-450 active site: Electron spin resonance spectroscopy as a probe of the hemin iron of cytochrome P-450: The influence of buffer composition, alcohols, and nitrogenous ligands

Abstract The electron spin resonance (esr) spectra of the low-spin form of hepatic microsomal cytochrome P -450 and of cytochrome P -450 isolated from Pseudomonas putida grown on d -camphor ( P -450 cam ) were studied in order to gain an understanding of the sensitivity of the hemin iron to changes in buffer. The shapes of the g x and g y esr signals of both the membrane-bound microsomal and soluble bacterial cytochromes P -450 were dependent upon buffer composition. With either system, the g x and g y signals were symmetric in some buffers and asymmetric in others. However, in potassium phosphate buffer, the esr spectra of low-spin cytochrome P -450 in microsomes isolated from phenobarbital (PB)- or 3-methylcholanthrene (3-MC-induced rats and cytochrome P -450 cam are similar with symmetric g x and g y signals. The esr spectrum of the low-spin form of cytochrome P -450 in isolated hepatocytes is similar to that of the microsomal and bacterial enzyme, again with a symmetric g x signal. The effects of alcohols and nitrogenous ligands on the esr spectrum of the low-spin form were also investigated. The data indicate that extreme care must be exercised when interpreting esr spectra with respect to possible cytochrome P -450 heterogeneity in the microsomal membrane. The conditions for studying substrate interactions with microsomal cytochrome P -450 must also take into account these changes in symmetry of the esr spectrum.

Dual wavelength/stopped flow spectrophotometry: Computer acquisition and analysis

Abstract The adaptation of a commercially available dual wavelength/stopped flow spectrophotometer for use with turbid samples is described. A minicomputer is used to collect and analyze the data, thereby facilitating these experiments. The stopped flow/computer combination has a dead time in the single wavelength mode of 3.5 msec. In the dual wavelength mode, accurate determinations can be made of the time course of reactions that have a t 1 2 of 50 msec or longer. The application of this stopped flow spectrophotometer to the measurement of cytochrome P-450 reductase activity in rat liver microsomes is described.

Metabolic Control of Cytochrome P-450 cam

Long before cytochrome P-450 was discovered, it was known that the metabolism of steroids was inhibited by CO (Ryan and Engel, 1957). It was a study of carbon monoxide inhibition of steroid metabolism by adrenal cortex which led to the discovery of the involvement of cytochrome P-450 in this reaction (Estabrook et al., 1963). Following this original observation, carbon monoxide inhibition of various monooxygenase reactions has been used in a number of laboratories to study the metabolism of these compounds. Philadelphia has been the site of a number of memorable experiments in the elucidation of the role of this enzyme in drug and steroid metabolism. Therefore, it is quite fitting that the subject of this paper be the control of cytochrome P-450 reactions and the role which carbon monoxide inhibition of cytochrome P-450 has played in obtaining an understanding of this control.

Dual wavelength/split beam spectrophotometry: Computer acquisition of absorbance spectra☆

Abstract The adaptation of a commercially available dual wavelength/split beam spectrophotometer for the real time acquisition by a minicomputer of absorbance spectra is described. The computer software and interface are designed so that: a) The normal operation of the spectrophotometer is unaltered; b) the adaptation of the spectrophotometer for computer acquisition of spectra is relatively simple and only a rudimentary knowledge of electronics and computer programming is required. The utility of this adaptation of a dual wavelength/split beam spectrophotometer for baseline correction, enhancement of the sensitivity of the spectrophotometer and subsequent analysis of recorded absorbance spectra is described.

An approach to the collection and manipulation of time-based data using the IBM PC and BASICA

With the advent of increasingly integrated, powerful and inexpensive digital electronics, relatively powerful computers have become available to the general public. Along with this technological boom there has been a concomitant increase in the availability of over-the-counter software packages which can be used by research scientists for program development. In the past, the development of computer programs for the collection of large amounts of time-based data was expensive and time consuming; however, the introduction of the current generation of 16-bit microcomputers and associated hardware and software packages has enabled investigators with only a rudimentary knowledge of computers and interfacing to begin to design programs. The schemes and algorithms, developed using BASICA on an IBM-Personal Computer, which are described in this article can serve other investigators as models for the assembly of their own programs for the collection, manipulation and plotting of time-based data. The incorporation of inexpensive computer graphics hardware and software, which provided a simple solution to the problem of analysis and presentation of large amounts of data, will also be discussed.

MECHANISM OF ACTION OF CYTOCHROME P-450: ROLE OF THE MICROSOMAL MEMBRANE

The observation and interpretation of temperature-dependent spin state changes associated with cytochrome P-450 have been the focus of numerous publications. The results presented in this paper clearly show that under these conditions: 1) there is no significant temperature-dependent spin state change of the hemin iron of cytochrome P-450 which does not involve reaction with substrate; and 2) solubility of the organic substrates in the microsomal membrane is an important factor in concentrating substrates in the environment of the enzyme and thus controlling the access of any particular substrate to the active site.