Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
MinireviewThe mystery of cytochrome P450 Compound I: A mini-review dedicated to Klaus Ruckpaul
Introduction
Cytochromes P450 (P450) represent a large superfamily of heme thiolate proteins which catalyze the conversion of a variety of chemically diverse compounds by insertion of an activated oxygen atom into an inert C-H bond [1]. The activated oxygen is formed at the so-called sixth iron coordination position of the protoporphyrin IX iron complex, which is the catalytic center surrounded by the protein of the enzyme and with the substrate located close to the oxygen (Fig. 1). The porphyrin complex is held in the protein matrix by coordinating the H-bonded, negatively charged sulfur atom of a cysteine to the iron and by salt links, diverse van der Waals contacts and H-bonded water molecules in the heme pocket [2]. The protein-bound porphyrin complex with the coordinated active oxygen is called Compound I, which is presumably the intermediate species which hydroxylates the substrate. When reading these last sentences, it might seem that the nature and reactivity of Compound I have been well characterized and the mechanism of its formation and of substrate conversion is completely discovered and understood. However, the large number of experiments and theoretical studies, reported over more than 45 years, has not lead to a final characterization of Compound I in P450.
It was during my PhD work in the 1980s that I heard from “Compound I” for the first time when I worked in the group of Klaus Ruckpaul in Berlin. He initiated the writing of a series of publications about P450 in the journal “Die Pharmazie,” volume 33, issues 6 and 7 (1978) where my part was to summarize thermodynamic aspects and ideas about the nature of the active oxygen species [3]. This series of publications was written with a view of the second Scientific Conference on “Cytochrome P-450: Structural and Functional Aspects” held in Eberswalde close to Berlin (July 9–13, 1978) which was organized by Klaus Ruckpaul [4], [5] and taken as basis for the book in 1984 about P450 edited by Klaus Ruckpaul and Horst Rein [6]. Ruckpaul and Rein continued in reviewing the progress in P450 research by functioning as editors of the book series “Frontiers in Biotransformation” starting with volume 1 in 1989 [7].
What was known about Compound I of P450 at that time when Ruckpaul's first book [6] appeared and what new discoveries have been made in the last 26 years? The number of publications about Compound I of P450 is too big to consider completely in this mini-review. Therefore, I will highlight only selected studies which I would qualify as key experiments or milestones for discovering the secrets of P450 Compound I. Comprehensive reviews appeared in the past [8], [9], [10], [11], [12], [13] and very recently [14], [15]. The conclusion from this review is that Compound I remains still a mystery and much efforts are needed to catch Compound I under experimental conditions which allow characterizing it with different spectroscopic techniques applied at the same sample.
Section snippets
Mechanisms of substrate hydroxylation and how the reactive oxygen species in P450 became assigned to Compound I—proposals in the 1980s
Ruckpaul [5], [6], Estabrook [16], Omura [17] and many others have described how P450 was discovered and which key experiments led to describe the main steps in the P450 catalyzed substrate conversions (Fig. 2). The main steps include (i) substrate binding to the Fe3+ complex of P450; (ii) first reduction to build up the Fe2+ state; (iii) binding of O2 to the Fe2+ heme, (iv) delivery of the second electron; (v) cleavage of the O-O bond of the iron-bound dioxygen; (vi) insertion of an oxygen
Hunting Compound I of P450—combined new and old approaches and suggestions
There is a general consensus in the P450 community to believe that Compound I, resulting from the heterolytic splitting of the O-O bond, does really exist and represents an iron–oxo species with iron in the Fe4+ state and the porphyrin with the cation radical in the π-system as described above for Suggestion 8. How could one prove this structural property?
The only way is to compare the spectroscopic properties and parameters of this presumed Compound I species with well studied reference
Apparently inconsistent results are obtained
Compound I, well characterized for CPO, HRP and other peroxidases to be a Fe4+-oxo porphyrin–π-cation radical complex, turned out to be a strange species in P450. The different methods (Fig. 4), used to catch and characterize this intermediate, revealed apparently inconsistent results. In the natural pathway, Compound I could not be detected. For the shunt pathway a UV-visible spectrum resembling that of CPO Compound I could be obtained only using the rapid-mixing stopped-flow technique.
Outlook
Reviewing the studies discussed above, one might think that Compound I in P450 appears to be a mystery. Indeed, over more than 45 years the P450 community tries to uncover the secrets of Compound I formation and mechanism of its action in substrate conversion. The knowledge has significantly increased although we still ask the same questions—does Compound I as Fe4+–oxo porphyrin–π-cation radical really exist and how would the oxygen atom be transferred to the C-H bond of the substrate. I
Acknowledgments
The diverse studies on cytochrome P450 were funded by the Deutsche Forschungsgemeinschaft grants Ju229/4-(1-3), Ju229/5-1, Sk35/3-(3-5) to C. Jung; Le 812/2-1 to F. Lendzian; Tr97/26-(1-3) to A.X. Trautwein; Schu1251/3-1 to V. Schünemann. The author thanks all former coworkers and students, in particular J. Contzen and M. Richter, for contributing to projects related to the subject of this review. Special gratitude goes to A. X. Trautwein, V. Schünemann and F. Lendzian for the fruitful
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