Structural and thermodynamic characterization of the adrenodoxin-like domain of the electron-transfer protein Etp1 from Schizosaccharomyces pombe.

The protein Etp1 of Schizosaccharomyces pombe consists of an amino-terminal COX15-like domain and a carboxy-terminal ferredoxin-like domain, Etp1(fd), which is cleaved off after mitochondrial import. The physiological function of Etp1(fd) is supposed to lie in the participation in the assembly of iron-sulfur clusters and the synthesis of heme A. In addition, the protein was shown to be the first microbial ferredoxin being able to support electron transfer in mitochondrial steroid hydroxylating cytochrome P450 systems in vivo and in vitro, replacing thereby the native redox partner, adrenodoxin. Despite a sequence similarity of 39% and the fact that fission yeast is a mesophilic organism, thermodynamic studies revealed that Etp1(fd) has a melting temperature more than 20°C higher than adrenodoxin. The three-dimensional structure of Etp1(fd) has been determined by crystallography. To the best of our knowledge it represents the first three-dimensional structure of a yeast ferredoxin. The structure-based sequence alignment of Etp1(fd) with adrenodoxin yields a rational explanation for their observed mutual exchangeability in the cytochrome P450 system. Analysis of the electron exchange with the S. pombe redox partner Arh1 revealed differences between Etp1(fd) and adrenodoxin, which might be linked to their different physiological functions in the mitochondria of mammals and yeast.

Kinetic and optical biosensor study of adrenodoxin mutant AdxS112W displaying an enhanced interaction towards the cholesterol side chain cleavage enzyme (CYP11A1).

In mammals, steroid hormones are synthesized from cholesterol that is metabolized by the mitochondrial CYP11A1 system leading to pregnenolone. The reduction equivalents for this reaction are provided by NADPH, via a small electron transfer chain, consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx). The reaction partners are involved in a series of transient interactions to realize the electron transfer from NADPH to CYP11A1. Here, we compared the ionic strength effect on the AdR/Adx and Adx/CYP11A1 interactions for wild-type Adx and mutant AdxS112W. Using surface plasmon resonance measurements, stopped flow kinetic investigations and analyses of the product formation, we were able to obtain new insights into the mechanism of these interactions. The replacement of serine 112 by tryptophan was demonstrated to lead to a dramatically decreased k (off) rate of the Adx/CYP11A1 complex, resulting in a four-fold decreased K (d) value and indicating a much higher stability of the complex involving the mutant. Stopped flow analysis at various ionic strengths and in different mixing modes revealed that the binding of reduced Adx to CYP11A1 seems to display the limiting step for electron transfer to CYP11A1 with pre-reduced AdxS112W being much more efficient than wild-type Adx. Finally, the dramatic increase in pregnenolone formation at higher ionic strength using the mutant demonstrates that the interaction of CYP11A1 with Adx is the rate-limiting step in substrate conversion and that hydrophobic interactions may considerably improve this interaction and the efficiency of product formation. The data are discussed using published structural data of the complexes.

The endogenous adrenodoxin reductase-like flavoprotein arh1 supports heterologous cytochrome P450-dependent substrate conversions in Schizosaccharomyces pombe.

Mitochondrial cytochromes P450 are essential for biosynthesis of steroid hormones, vitamin D and bile acids. In mammals, the electrons needed for these reactions are provided via adrenodoxin and adrenodoxin reductase (AdR). Recently, Schizosaccharomyces pombe was introduced as a new host for the functional expression of human mitochondrial steroid hydroxylases without the coexpression of their natural redox partners. This fact qualifies S. pombe for the biotechnological production of steroids and for application as inhibitor test organism of heterologously expressed cytochromes P450. In this paper, we present evidence that the S. pombe ferredoxin reductase, arh1, and ferredoxin, etp1fd provide mammalian class I cytochromes P450 with reduction equivalents. The recombinant reductase showed an unusual weak binding of flavin adenine dinucleotide (FAD), which was mastered by modifying the FAD-binding region by site-directed mutagenesis yielding a stable holoprotein. The modified reductase arh1_A18G displayed spectroscopic characteristics similar to AdR and was shown to be capable of accepting electrons with no evident preference for NADH or NADPH, respectively. Arh1_A18G can substitute for AdR by interacting not only with its natural redox partner etp1fd but also with the mammalian homolog adrenodoxin. Cytochrome P450-dependent substrate conversion with all combinations of the mammalian and yeast redox proteins was evaluated in a reconstituted system.

Stripping down the mitochondrial cholesterol hydroxylase system, a kinetics study.

The origin of steroid hormones in mammals is cholesterol that is metabolized by the mitochondrial CYP11A1 system. The cytochrome P450 is fed with reduction equivalents via a small electron transfer chain consisting of NADPH, adrenodoxin reductase, and adrenodoxin. Though the redox behavior of the individual protein components has been studied previously, the kinetics of the system in its entirety has not yet been analyzed. In this study we combine surface plasmon resonance experiments to determine the binding constants for the different pairs of redox partners with measurements of the pre-steady-state kinetics of the different reaction steps of this system and steady-state kinetics. We could correlate the individual protein-protein interactions with the effect of distinct reduction-oxidation steps on the overall catalytic activity of the CYP11A1 system. For the first time, we were able to follow the reduction of each of the protein components of this system within one measurement when we mixed all oxidized protein components with NADPH. These measurements allowed the determination of the individual apparent rate constants for the reduction of all three proteins involved. In addition, variation of the ionic strength in these experiments revealed different optimum salt concentrations for the reduction of adrenodoxin reductase and adrenodoxin, respectively, and unraveled dramatically changing reduction rates of CYP11A1 by adrenodoxin.

The adrenodoxin-like ferredoxin of Schizosaccharomyces pombe mitochondria.

The single mitochondrial type I [2Fe-2S] ferredoxin of the fission yeast Schizosaccharomyces pombe is produced as the carboxy terminal part of the electron-transfer-protein 1 (etp1) and cleaved off during mitochondrial import [Biochemistry 41 (2002) 2311-2321]. The UV/Vis (UV-visible) spectrum of the purified recombinant ferredoxin domain (etp1(fd)) expressed in Escherichia coli is similar to those of bovine Adx in the oxidized as well as in the reduced state. EPR (electronic paramagnetic resonance) studies revealed a correctly incorporated iron-sulfur cluster of the axial type. The redox potential of this protein was determined to be -353 mV, which is considerably lower than that of adrenodoxin (Adx, -273 mV). Several lines of evidence indicate that the protein forms dimers under physiological and denaturating conditions. Interestingly, the fission yeast ferredoxin could be shown to be active as an electron carrier in heterologous redox systems. It is able to transfer electrons to horse heart cytochrome c and to bovine cytochromes P450(scc) (CYP11A1) and P450(11 beta) (CYP11B1), thereby receiving electrons from bovine NADPH-dependent Adx reductase. The kinetics of substrate conversion in the etp1(fd)-supported CYP11A1 and CYP11B1-dependent systems mediated was studied.

Effect of fermentation on the starch digestibility, resistant starch and some physicochemical properties of sorghum flour.

The effect of natural fermentation of Tabat sorghum cultivar (Sorghum bicolor L. Moench) at 37 degrees C for up to 36 h on pH, titratable acidity, starch digestibility, resistant starch and total starch was studied. The pH of the fermenting dough decreased sharply with a concomitant increase in the titratable acidity. In vitro starch digestibility markedly increased as a result of fermentation, while resistant starch and total starch decreased. Results showed that iodine absorption capacity increased during fermentation. Fermented sorghum had more soluble starch and swelling power at 100 degrees C than at 85 degrees C.

Bacterial (CYP101) and mitochondrial P450 systems-how comparable are they?

The bacterial CYP101 system and mitochondrial P450 systems show high similarity. Both systems contain the same protein components, a FAD containing reductase, a ferredoxin of the [2Fe2S] type, and a cytochrome P450. At a first glance they seem to be comparable but there are considerable differences among both proteins. Thus, the ferredoxin components of the two systems display significant structural homology but cannot substitute for each other in functional assays. Going into more detail, pronounced differences between the two systems that affect their biological functions are found.

Insights into the design of a hybrid system between Anabaena ferredoxin-NADP+ reductase and bovine adrenodoxin.

The opportunity to design enzymatic systems is becoming more feasible due to detailed knowledge of the structure of many proteins. As a first step, investigations have aimed to redesign already existing systems, so that they can perform a function different from the one for which they were synthesized. We have investigated the interaction of electron transfer proteins from different systems in order to check the possibility of heterologous reconstitution among members of different chains. Here, it is shown that ferredoxin-NADP+ reductase from Anabaena and adrenodoxin from bovine adrenal glands are able to form optimal complexes for thermodynamically favoured electron transfer reactions. Thus, electron transfer from ferredoxin-NADP+ reductase to adrenodoxin seems to proceed through the formation of at least two different complexes, whereas electron transfer from adrenodoxin to ferredoxin-NADP+ reductase does not take place due because it is a thermodynamically nonfavoured process. Moreover, by using a truncated adrenodoxin form (with decreased reduction potential as compared with the wild-type) ferredoxin-NADP+ reductase is reduced. Finally, these reactions have also been studied using several ferredoxin-NADP+ reductase mutants at positions crucial for interaction with its physiological partner, ferredoxin. The effects observed in their reactions with adrenodoxin do not correlate with those reported for their reactions with ferredoxin. In summary, our data indicate that although electron transfer can be achieved in this hybrid system, the electron transfer processes observed are much slower than within the physiological partners, pointing to a low specificity in the interaction surfaces of the proteins in the hybrid complexes.

Functional expression of human mitochondrial CYP11B2 in fission yeast and identification of a new internal electron transfer protein, etp1.

Mitochondrial cytochrome P450 enzymes play a crucial role in the steroid biosynthesis in human adrenals, catalyzing regio- and stereospecific hydroxylations. In search of a new model system for the study of these enzymes, we expressed the human CYP11B2 (aldosterone synthase, P450(aldo)) in fission yeast Schizosaccharomyces pombe. Analysis of the subcellular localization of the P450 enzyme by Western blot analysis, fluorescence microscopy, and electron microscopy demonstrated that the mitochondrial localization signal of the human protein is functional in S. pombe. The transformed yeasts show the inducible ability to convert in vivo considerable amounts of 11-deoxycortisol to cortisol and 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone, and aldosterone, respectively. Although in mammalian cells, mitochondrial steroid hydroxylases depend for their activity on an electron transport chain that consists of two proteins, adrenodoxin and adrenodoxin reductase, no coexpression of these proteins is needed for efficient substrate conversion by intact fission yeast cells. Searching the fission yeast genome for adrenodoxin homologues, a gene was identified that codes for a protein with an amino terminal domain homologous to COX15 of Saccharomyces cerevisiae and a carboxy terminal ferredoxin domain. It was found that overexpression of this gene significantly enhances steroid hydroxylase activity of CYP11B2 expressing fission yeast cells. Moreover, the bacterially expressed ferredoxin domain of this protein can replace adrenodoxin in a reconstituted steroid hydroxylation assay and transfer electrons from adrenodoxin reductase to a mammalian or a bacterial cytochrome P450. Therefore, we suggest to name this protein etp1 (electron-transfer protein 1).