Gabriele A. Ziegler, Clemens Vonrhein, Georg E. Schulz, and *Israel Hanukoglu
Institut fur Organische Chemie und Biochemie, Albert-Ludwigs-Universitat, 79104 Freiburg im Breisgau, Germany, and *Department of Chemical Engineering and Biotechnology, College of Judea and Samaria, 44837 Ariel, Israel.
(This review should be referenced as:
Ziegler G. A., Vonrhein C., Schulz G. E., and Hanukoglu I. Structures of adrenodoxin reductase and adrenodoxin support shuttle mechanism of electron transfer in mitochondrial P450 systems. In: Molecular Steroidogenesis (M. Okamoto, Y. Ishimura, H. Nawata, eds.), Universal Academy Press, Tokyo, Japan, pp. 61-64, 2000.)
- Abstract
- Introduction
- Kinetic models of electron transfer in mitochondrial P450 systems
- Identification of the amino acid residues involved in adrenodoxin binding
- References
Abstract
Mitochondrial P450 type enzymes are generally involved in the metabolism of cholesterol derived steroidal compounds. Each P450 catalyzed reaction requires molecular oxygen and two electrons donated by NADPH. The electrons of NADPH are transferred to P450 by an electron transfer system that includes a specific flavoprotein, adrenodoxin reductase, and an iron-sulfur protein, adrenodoxin. The crystal structure of adrenodoxin reductase we recently determined revealed that this enzyme is a kidney shaped protein with two distinct faces, a basic face and an acidic face. Lysine residues suggested to be involved in adrenodoxin binding protrude on the basic side. Manual docking of adrenodoxin and reductase molecules side by side supports that adrenodoxin residues Asp-76 and Asp-79 could be involved in specific binding to reductase by electrostatic interactions. At the modeled orientation, the distance for electron transfer from FAD to 2Fe-2S center of adrenodoxin is about 16 ?. These findings and other reviewed evidence, support the kinetic model that adrenodoxin functions as a mobile electron shuttle, and not within a static ternary complex of reductase-adrenodoxin and P450.
Introduction
Mitochondrial P450 type enzymes catalyze essential steps in the biosynthesis of steroid hormones and the metabolism of cholesterol derived steroidal compounds. The reactions catalyzed by these enzymes include cholesterol conversion to pregnenolone, 11β- and 18- hydroxylation reactions in steroid biosynthesis, C-27 hydroxylation of cholic acid in bile acid metabolism, and 1a and 24 hydroxylations of 25-OH-vitamin D (1-6). These reactions are catalyzed by specific P450s and follow the usual monooxygenation stoichiometry:
R-H + NADPH + H+ + O2 → R-OH + NADP+ + H2O
The two electrons donated by NADPH are transferred to P450 via two electron transfer proteins, adrenodoxin reductase, which is an FAD containing flavoenzyme, and adrenodoxin, which is a [2Fe-2S] ferredoxin type iron-sulfur protein (1-6). FAD of adrenodoxin reductase accepts two electrons from NADPH, and these are transferred one at a time to adrenodoxin, which is a one-electron carrier. In these respects the mitochondrial P450 electron transfer system is similar to that of some bacterial P450's such as P450cam from Pseudomonas putida that includes a ferredoxin reductase and a ferredoxin (putidaredoxin).
All three proteins of mitochondrial P450 systems are located on the matrix side of the inner mitochondrial membrane (3). Whereas the mitochondrial P450s are tightly bound to the membrane, the electron transfer proteins are soluble in the matrix. All three proteins are encoded as larger precursors, and their signal peptides are cleaved during transfer into mitochondria (Table 1). In contrast to the multiplicity of P450 forms, both adrenodoxin reductase, and adrenodoxin, are encoded by one or two similar nuclear genes in all animal species (Table 1). These genes are expressed in all human tissues examined. Thus, the electron transfer proteins are not specific to individual P450s and serve as electron donors for different cytochromes P450 in different tissues.
Recently we determined the first crystal structure of adrenodoxin reductase (7). The structure of adrenodoxin has been also recently elucidated (8). Here we review studies on the interactions of these electron transfer proteins in the light of their structures.
| Species | SWISSPROT code | Prepeptide | Signal peptide | Mature peptide Length MW | ||
|---|---|---|---|---|---|---|
| Adrenodoxin Red. | Human | adro_human | 491 | 32 | 459 | 49967 |
| Bovine | adro_bovin | 492 | 32 | 460 | 50296 | |
| Rat | adro_rat | 494 | 34 | 460 | 50316 | |
| Mouse | adro_mouse | 494 | 34 | 460 | 50128 | |
| Yeast | adro_yeast | 493 | ||||
| Adrenodoxin | Human | adx_human | 184 | 60 | 124 | 13561 |
| Bovine | adx1_bovin | 186 | 58 | 128 | 14048 | |
| Sheep | adx_sheep | N.D. | ||||
| Pig | adx_pig | 186 | 58 | 128 | 14012 | |
| Rat | adx_rat | 188 | 64 | 124 | 13588 | |
| Mouse | adx_mouse | 188 | 64 | 124 | 13617 | |
| Chick | adx_chick | N.D. | 124 | 13558 | ||
Kinetic models of electron transfer in mitochondrial P450 systems
Previous studies on the mitochondrial P450 systems have suggested two distinct models for electron transfer from the reductase to the P450: 1) A dynamic shuttle model: This model hypothesizes that adrenodoxin forms separate binary complexes with adrenodoxin reductase and P450 and that it shuttles between these two proteins, as it accepts an electron from the reductase and transfers it to the P450, and then rebinds to reductase to repeat the same function. 2) A static ternary complex model: This model hypothesizes that reductase, adrenodoxin and P450 can form a ternary complex including all three proteins of the system.
The dynamic shuttle model was first proposed by studies that showed that a 1:1:1 ternary complex of adrenodoxin reductase, adrenodoxin, and P450 does not form under equilibrium or steady state turnover conditions (5, 9-11). The activities of mitochondrial P450s show Michaelis-Menten dependence on free reduced adrenodoxin (9-11). These kinetic studies also showed that reductase competes with P450 for binding to adrenodoxin. The structural implication of these studies was that both reductase and mitochondrial P450s have overlapping binding sites on adrenodoxin, resulting in competitive binding to adrenodoxin.
Determination of the concentrations of adrenodoxin reductase, adrenodoxin and P-450 in the adrenal cortex revealed that the molar amounts of reductase is about one tenth of the amount of the P450 in the mitochondria (12). These observations also argued against the static model of ternary complexes. Based on the kinetic studies noted above, electron transport system would function most efficiently if most of the adrenodoxin molecules are maintained in reduced form and unbound to reductase. These biological design specifications are apparently met by the characteristics of the system: 1. A relatively low concentration of reductase to minimize the competition of reductase with P450 for binding to adrenodoxin, 2. A much faster turnover rate of adrenodoxin reduction by reductase than the rate of adrenodoxin oxidation by P450 so that the low concentrations of reductase suffice for the system, and 3. Redox equilibria that favor dissociation of adrenodoxin from reductase after reduction, and binding of adrenodoxin to P450-substrate complex.
A putative ternary complex was first reported only under equilibrium conditions (13). These results were interpreted differently, refuting the conclusion of a ternary complex (see discussion in ref. 5). Another suggested evidence for ternary complex formation is based on chemical cross-linking studies (14). Yet, in such complexes the proteins may be cross-linked at sites different from the high affinity binding sites identified in equilibrium binding studies. Harikrishna et al., (15) designed and expressed in COS cells fused proteins including two or three components of the mitochondrial P450 system at different orientations. Cells expressing some of these constructs showed greater activity than cells expressing each component from separate cotransfected vectors. It should be noted however, that each construct might not necessarily function on its own transferring electrons within the fused multi-protein complex, as its individual components may interact with other fused proteins in the mitochondria. Thus, the function of these fused proteins does not exclude the shuttle mechanism.
Identification of the amino acid residues involved in adrenodoxin binding
The association of adrenodoxin with reductase and P450 is dependent on ionic interactions (2, 11, 16). Hydrophobic interactions do not appear to play a role in adrenodoxin- P450 complex formation as the Kd of the complex was unaffected by an uncharged detergent (11). These findings suggested that complementary charged residues are involved in the specific complex formations. Indeed, site directed mutations of human adrenodoxin reductase and adrenodoxin provided evidence that Arg-239 and Arg-243 of adrenodoxin reductase interact directly with Asp-76 and Asp-79 of adrenodoxin (for references see Table 2).
The crystal structure of bovine adrenodoxin reductase we recently determined revealed that this enzyme is a kidney shaped protein with two distinct faces, a neutral-basic face and an acidic face (15). Lys-240 and Lys-244 (bovine sequence numbering) suggested to be involved in adrenodoxin binding protrude on the basic side. Thus, the crystal structure presents a picture that is consistent with the site-directed mutagenesis studies (Table 2).
| Protein | Residues essential for binding | Exposed on Protein surface | References |
|---|---|---|---|
| Adrenodoxin | |||
| Adrenodoxin reductase | Arg-240 | Yes | 7, 17-20 |
| Arg-244 | |||
| Yes | |||
| Adrenodoxin reductase | |||
| Adrenodoxin | Asp-76 | Yes | 8, 17-20 |
| Asp-79 | Yes | ||
| P450scc | |||
| Thr-54 | Yes | 8, 17-22 | |
| Asp-76 | Yes | ||
| Asp-79 | Yes | ||
| Tyr-82 | Yes | ||
| C-terminal | |||
| Adrenodoxin | |||
| P450scc | Lys-377 | 23 | |
| Lys-381 | |||
| P450c27 | Lys-354 | 24 | |
| Lys-358 | |||
| Arg-362 | 25 | ||
| Arg-418 | 24 |
Site directed mutation of adrenodoxin at Asp-76 and Asp-79 showed that these residues are essential also for adrenodoxin binding to P450scc (Table 2). Similar to reductase, the crystal structure of adrenodoxin also supports the earlier site directed mutation studies, as the side chains of these aspartates are exposed on the surface of adrenodoxin. Independent studies on two different mitochondrial P450's, P450scc and P450c27 have identified a cluster of basic lysine and arginine residues on these enzymes (Table 2). Thus, it appears that both adrenodoxin reductase and P450 utilize similar structural features to bind to the same acidic aspartates on adrenodoxin. Overall these observations support the conclusion of earlier kinetic studies that the binding sites of these two enzymes on adrenodoxin overlap. Yet, the differential effects on interactions with reductase and P450 (listed in Table 2) indicate that the sites are not identical.
Manual docking of adrenodoxin and reductase show that a specific interaction between Lys-240 and Lys-244 of reductase with Asp-76 and Asp-79 of adrenodoxin is possible (7). At the modeled orientation, the distance for electron transfer from FAD to 2Fe-2S center of adrenodoxin is about 16 ?. These findings and other reviewed evidence, support the kinetic model that adrenodoxin functions as a mobile electron shuttle, and not within a static ternary complex of reductase-adrenodoxin and P450. Yet, one of the important pieces that are missing to complete the puzzle is the elucidation of the crystal structure of a mitochondrial P450. After the resolution of the binding sites, the next problem that awaits us is the identification of the pathway(s) of electron within and between the active sites of these proteins.
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