No abstract

Mammalian adrenodoxin (ferredoxin 1; Fdx1) is essential for the synthesis of various steroid hormones in adrenal glands. As a member of the [2Fe-2S] cluster-containing ferredoxin family, Fdx1 reduces mitochondrial cytochrome P450 enzymes, which then catalyze; e.g., the conversion of cholesterol to pregnenolone, aldosterone, and cortisol. The high protein sequence similarity between Fdx1 and its yeast adrenodoxin homologue (Yah1) suggested that Fdx1, like Yah1, may be involved in the biosynthesis of heme A and Fe/S clusters, two versatile and essential protein cofactors. Our study, employing RNAi technology to deplete human Fdx1, did not confirm this expectation. Instead, we identified a Fdx1-related mitochondrial protein, designated ferredoxin 2 (Fdx2) and found it to be essential for heme A and Fe/S protein biosynthesis. Unlike Fdx1, Fdx2 was unable to efficiently reduce mitochondrial cytochromes P450 and convert steroids, indicating that the two ferredoxin isoforms are highly specific for their substrates in distinct biochemical pathways. Moreover, Fdx2 deficiency had a severe impact, via impaired Fe/S protein biogenesis, on cellular iron homeostasis, leading to increased cellular iron uptake and iron accumulation in mitochondria. We conclude that mammals depend on two distinct mitochondrial ferredoxins for the specific production of either steroid hormones or heme A and Fe/S proteins.adrenodoxin | cytochrome P450 | iron | iron-sulfur cluster | IRP1

Adrenodoxin is an iron‐sulfur protein that belongs to the broad family of the [2Fe‐2S]‐type ferredoxins found in plants, animals and bacteria. Its primary function as a soluble electron carrier between the NADPH‐dependent adrenodoxin reductase and several cytochromes P450 makes it an irreplaceable component of the steroid hormones biosynthesis in the adrenal mitochondria of vertebrates. This review intends to summarize current knowledge about structure, function, and biochemical behavior of this electron transferring protein. We discuss the recently solved first crystal structure of the vertebrate‐type ferredoxin, the truncated adrenodoxin Adx(4‐108), that offers the unique opportunity for better understanding of the structure‐function relationships and stabilization of this protein, as well as of the molecular architecture of [2Fe‐2S] ferredoxins in general. The aim of this review is also to discuss molecular requirements for the formation of the electron transfer complex. Essential comparison between bacterial putidaredoxin and mammalian adrenodoxin will be provided. These proteins have similar tertiary structure, but show remarkable specificity for interactions only with their own cognate cytochrome P450. The discussion will be largely centered on the protein‐protein recognition and kinetics of adrenodoxin dependent reactions. Proteins 2000;40:590–612. © 2000 Wiley‐Liss, Inc.

In the general view of protein-complex formation, a transient and dynamic encounter complex proceeds to form a more stable, well-defined, and active form. In weak protein complexes, however, the encounter state can represent a significant population of the complex. The redox proteins adrenodoxin (Adx) and cytochrome c (C c) associate to form such a weak and short-lived complex, which is nevertheless active in electron transfer. To study the conformational freedom within the protein complex, the native complex has been compared to a cross-linked counterpart by using solution scattering and NMR spectroscopy. Oligomerization behavior of the native complex in solution revealed by small-angle X-ray scattering indicates a stochastic nature of complex formation. For the cross-linked complex, interprotein paramagnetic effects are observed, whereas for the native complex, extensive averaging occurs, consistent with multiple orientations of the proteins within the complex. Simulations show that C c samples about half of the surface area of adrenodoxin. It is concluded that the complex of Adx/C c is entirely dynamic and can be considered as a pure encounter complex.