Adrenodoxin--a versatile ferredoxin.

Mammalian adrenodoxin (Adx) has been known for many years as an essential electron mediator in mitochondrial cytochrome P450 systems. Because of its ability to support several cytochrome P450 enzymes, it is involved not only in adrenal steroid hormone biosynthesis but also in vitamin D and bile acid metabolism. Recently, Adx is increasingly gaining attention because of its potential for pharmaceutical industry and biotechnology. With human cytochromes P450 becoming important drug targets, suitable Adx-based screening systems have to be developed to test putative new drugs. Moreover, in artificial systems, Adx has been shown to functionally interact with diverse bacterial cytochromes P450 catalyzing a variety of chemically interesting reactions. Putative biotechnological applications of such Adx-containing reconstituted systems are discussed.

Functional characterization of Fdx1: evidence for an evolutionary relationship between P450-type and ISC-type ferredoxins.

Ferredoxins are ubiquitous proteins with electron transfer activity involved in a variety of biological processes. In this work, we investigated the characteristics and function of Fdx1 from Sorangium cellulosum So ce56 by using a combination of bioinformatics and of biochemical/biophysical approaches. We were able to experimentally confirm a role of Fdx1 in the iron-sulfur cluster biosynthesis by in vitro reduction studies with cluster-loaded So ce56 IscU and by transfer studies of the cluster from the latter protein to apo-aconitase A. Moreover, we found that Fdx1 can replace mammalian adrenodoxin in supporting the activity of bovine CYP11A1. This makes S. cellulosum Fdx1 the first prokaryotic ferredoxin reported to functionally interact with this mammalian enzyme. Although the interaction with CYP11A1 is non-physiological, this is-to the best of our knowledge-the first study to experimentally prove the activity of a postulated ISC-type ferredoxin in both the ISC assembly and a cytochrome P450 system. This proves that a single ferredoxin can be structurally able to provide electrons to both cytochromes P450 and IscU and thus support different biochemical processes. Combining this finding with phylogenetic and evolutionary trace analyses led us to propose the evolution of eukaryotic mitochondrial P450-type ferredoxins and ISC-type ferredoxins from a common prokaryotic ISC-type ancestor.

Adrenodoxin: the archetype of vertebrate-type [2Fe-2S] cluster ferredoxins.

Adrenodoxin is probably the best characterized member of the vertebrate-type [2Fe-2S]-cluster ferredoxins. It has been in the spotlight of scientific interest for many years due to its essential role in mammalian steroid hormone biosynthesis, where it acts as electron mediator between the NADPH-dependent adrenodoxin reductase and several mitochondrial cytochromes P450. In this review we will focus on the present knowledge about protein-protein recognition in the mitochondrial cytochrome P450 system and the modulation of the electron transfer between Adx and its redox partners, AdR and CYP(s). We also intend to point out the potential biotechnological applications of Adx as a versatile electron donor to different cytochromes P450, both in vitro and in vivo. Finally we will address the comparison between the mammalian cytochrome P450-associated adrenodoxin and ferredoxins involved in iron-sulfur-cluster biosynthesis. Despite their different functions, these proteins display an amazing similarity regarding their primary sequence, tertiary structure and biophysical features.

ThnY is a ferredoxin reductase-like iron-sulfur flavoprotein that has evolved to function as a regulator of tetralin biodegradation gene expression.

Previous genetic studies in Sphingomonas macrogolitabida strain TFA have established that expression of genes involved in tetralin biodegradation (thn genes) requires the function of the LysR type activator ThnR and also ThnY. Sequence comparison indicated that ThnY is homologous to bacterial oxygenase-coupled NAD(P)H-dependent ferredoxin reductases. However, ThnY showed substitutions in highly conserved positions of the pyridine nucleotide binding domain of these ferredoxin reductases. ThnY expression is co-regulated with all other genes required for tetralin biodegradation, and presumably thnY is part of the thnCA3A4RY operon. ThnY has been purified, and its biochemical and functional properties were characterized. ThnY was found to be a monomeric orange-brown iron-sulfur flavoprotein (estimated mass of 37,000 Da) containing one non-covalently attached flavin adenine dinucleotide and one plant type ferredoxin 2Fe-2S cluster. It can be efficiently reduced by dithionite, but reduction by pyridine nucleotides was very poor. Consistently, ThnY-dependent reduction of cytochrome c, ferricyanide, or 2,6-dichlorophenolindophenol using NAD(P)H as the electron donor was undetectable or very weak. The addition of ThnY to electrophoretic mobility shift assays containing ThnR and a probe bearing two thn divergent promoters resulted in a 3-fold increase in protein-DNA complex formation affinity, which indicates that ThnY directly promotes thn transcription activation by ThnR.

Genome mining in Sorangium cellulosum So ce56: identification and characterization of the homologous electron transfer proteins of a myxobacterial cytochrome P450.

Myxobacteria, especially members of the genus Sorangium, are known for their biotechnological potential as producers of pharmaceutically valuable secondary metabolites. The biosynthesis of several of those myxobacterial compounds includes cytochrome P450 activity. Although class I cytochrome P450 enzymes occur wide-spread in bacteria and rely on ferredoxins and ferredoxin reductases as essential electron mediators, the study of these proteins is often neglected. Therefore, we decided to search in the Sorangium cellulosum So ce56 genome for putative interaction partners of cytochromes P450. In this work we report the investigation of eight myxobacterial ferredoxins and two ferredoxin reductases with respect to their activity in cytochrome P450 systems. Intriguingly, we found not only one, but two ferredoxins whose ability to sustain an endogenous So ce56 cytochrome P450 was demonstrated by CYP260A1-dependent conversion of nootkatone. Moreover, we could demonstrate that the two ferredoxins were able to receive electrons from both ferredoxin reductases. These findings indicate that S. cellulosum can alternate between different electron transport pathways to sustain cytochrome P450 activity.