The role of post-translational modifications in electron-transfer reactions remains mostly obscure. Phosphorylation is known to alter the functionality of some proteins by inducing conformational changes.70
Previous studies indicate that phosphorylation plays an active role in redox signaling that regulates energy transfer in chloroplasts,71
thus providing an example of signal transduction where electron-transfer reactions initiate phosphorylation and results in the regulation of the amount of harvested light by those organelles. The inverse process, namely the regulation of ET by phosphorylation, seems plausible. We have investigated the possible influences of phosphorylation on the adrenal mitochondrial steroid hydroxylating system. Here, electrons are being transferred from an NADPH-dependent reductase (AdR) to a [2Fe–2S] cluster ferredoxin (Adx) and then to a cytochrome P450 that catalyzes steroid hydroxylation.22
The overall rate-limiting step in steroidogenesis is the formation of pregnenolone from cholesterol which is catalyzed by CYP11A1.18b
The rate-limiting step in this electron-transfer chain, however, is the interaction between the ferredoxin and the cytochrome.19,36
Therefore, it was of interest to analyze the influences of phosphorylation on the interaction between Adx and cytochrome P450scc
Previous studies revealed that a homologous adrenodoxin protein from rat liver is phosphorylated by PKA72,73
and that bovine Adx is the only component of the mitochondrial steroid hydroxylating system that can be readily phosphorylated by CK2, a ubiquitous and highly conserved Ser and Thr kinase.1
The residue that is phosphorylated by this kinase, Thr-71, is located in the interaction domain of this ferredoxin that represents the primary region for redox partner recognition.23,74
This primary interaction region is more flexible than the core domain of the protein, and this conformational flexibility may be relevant for the interaction of Adx with its redox partners. Therefore, phosphorylation of Thr-71 could alter its functionality.
Phosphorylation of T71 (or substitution of this Thr by Glu) increases the negative charge of the surface interaction region of Adx. Since the recognition between Adx and its redox partners is mainly based on electrostatic interactions and the dipole moment in Adx is considered to contribute to the docking kinetics of Adx and its redox partners,22,75
additional negative charge could enhance complex formation. In the present studies, this hypothesis is confirmed by optical biosensor experiments as well as by the KA
values extracted from stopped-flow experiments. These experiments reproducibly () indicate an enhanced association constant toward CYP11A1 for Adx-T71E and phosphorylated Adx compared to WT-Adx, but there appears to be no effect on the association with AdR. Comparable results for interactions of Adx with CYP11A1 are obtained from the mean residence times extracted from BD simulations in . The differential effect of these charge alterations at residue T71 on AdR and CYP11A1 association supports the idea that the binding sites on Adx for its interaction partners are overlapping rather than identical, as previously postulated.22
In the steady-state approximation for the donor–acceptor complex concentration, the pseudo-first-order ET rate is
when one reactant is present in excess.76
In the saturation regime, the quasi-unimolecular rate is k1
, denoted kobsd,max
in the tables, obtained by fitting the experimental data to eq 3
. The bimolecular rate associated with interprotein ET (in the steady-state approximation) and when [X] is sufficiently small is
If ET is slower than complex dissociation, the bimolecular rate reaches the activation limit of k2
, and when ET is fast, the reaction is diffusion limited, k2
. The Brownian dynamics simulations have the ability to span the two mechanistic regimes without assuming either limiting behavior. Generalizations of this description that take explicit account of multiple donor–acceptor binding geometries appear in ref 15
. The slowness of the kinetics measured here suggests activation limited kinetics.
Stopped-flow kinetic analysis () shows a slightly enhanced (1.2-fold increase) CYP11A1 reduction rate with mutant Adx-T71E compared to WT, and the rate constants calculated from BD simulations produced the same trend. In contrast to all other Adx species, phosphorylated Adx displays biphasic reaction kinetics ( and ). These biexponential kinetics could indicate that reduced phosphorylated Adx is capable of binding to different regions of the cytochrome and that alternate ET pathways might be favored with the insertion of the phosphate group. Since the amplitude of the first rapid phase is greater (~ 60%) than that of the second phase (~ 40%), which also includes the reaction of the small percentage of unphosphorylated Adx present in the sample, the complex leading to faster ET kinetics could be thermodynamically favored over the conformation that resembles the complex conformation preferred by the other Adx species. The BD trajectory profiles in suggest that the phosphorylated Adx may preferentially associate with CYP11A1 in a different binding region than the WT-protein and the mutants, Adx-T71E and Adx-T71V. This secondary region on the CYP11A1 surface contains many polar and basic groups, which appear to attract the phosphate group and thus provide a more efficient electron-transfer pathway. By combining the information obtained from these docking profiles with the rate constants extracted from the stopped-flow experiments, it can be inferred that the slower second phase for the biexponential reduction of CYP11A1 by phosphorylated Adx corresponds to electron transfer at the binding site that WT-Adx, Adx-T71E, and Adx-T71V all seem to prefer, and the first fast phase corresponds to stronger binding to the additional electron-transfer site by the phosphorylated protein.
Of the two possible binding regions on the CYP11A1 surface, phosphorylated Adx appears to bind preferentially to the region near Lys-193, while all other species appear to bind preferentially to the region near residue Lys-405. As indicated by the biexponential stopped-flow kinetics, phosphorylated Adx is also capable of binding to the region near Lys-405 with an electron-transfer rate in the range observed for the other Adx species. Since the other Adx species seem to be capable as well of binding to the region around Lys-193, it may be postulated that the complex configuration involving Lys-193 may require a reorientation of the Adx species before ET is enabled, but that phosphorylated Adx may not require reorientation as has been proposed for other Adx species.37
This assumption may provide an explanation for the observation that WT-Adx and the two Adx mutants preferentially bind to the region near residue Lys-405 and additionally provides an explanation for the biexpo-nential behavior of the phosphorylated species. Since phospho-rylated Adx displays biexponential electron-transfer kinetics, we suggest that the phosphorylation of T71 increases the efficiency of the electron-transfer pathway of this second complex configuration.
These studies also indicate that the substitution of a Thr residue by Glu does not fully simulate phosphorylation but rather approximates a permanent phosphorylated state. The differences observed for the phosphorylated Adx and the positive control, Adx-T71E, could be caused by minor structural changes indicated in . In addition, a recent 31
P NMR study of the protonation state of o
-phosphoserine showed that the pKa
value for the second proton of the phosphate is approximately 5.9,77
so it can be hypothesized that phosphothreonine is also predominantly double deprotonated at pH 7.4. Therefore, a phosphorylation can introduce two additional negative charges and hence strengthen electrostatic interactions with the more basic region around Lys-193, whereas the substitution of Thr by Glu only introduces a single negative charge. This charge interaction could be involved in fine-tuning the orientation of phosphorylated Adx with CYP11A1 in this region. It is noteworthy that the mutant Adx-T71E exhibited a slightly increased ET rate and a significant increase in the binding affinity, indicating that this charge insertion enhances the ET in the region around Lys-405. The observation that both T71 mutants display differences in the docking profile compared to WT-Adx points toward an important role of the hydroxyl group of Thr-71 for the binding behavior of Adx toward CYP11A1. However, the fact that the mutant Adx-T71E preferentially associated with the region around residue Lys-405 indicates that the insertion of the phosphate group and hence two negative charges seems to facilitate the binding to the region around Lys-193, enabling a fast and efficient ET (which probably requires the above proposed fine-tuning mechanism). Moreover, the observed differences between the phosphorylated Adx and the T71E mutant could also be caused by the insertion of low-lying phosphate orbitals that directly enhance donor–acceptor interactions as well as stabilize the complex of phosphorylated Adx with the region around residue Lys-193 of CYP11A1.
The results presented here raise the question of whether phosphorylation of Adx at position T71 is physiologically relevant or not. Recently published in vitro and in vivo data indicate that phosphorylation of Adx at Thr-71 leads only to an increase in the interaction with CYP11A1 and not with CYP11B1: the other natural interaction partner of Adx. CYP11A1 catalyzes the initial rate-limiting step of steroid biosynthesis, namely the side chain cleavage reaction that leads to the conversion of cholesterol to pregnenolone, which is the precursor molecule for all steroid hormones (for review, see ref 18b
). Bovine CYP11B1, on the other hand, is involved in a series of hydroxylation reactions that result in the formation of cortisol and aldosterone, respectively (for review, see ref 20
). A differentially altered interaction of phosphorylated Adx with CYP11A1 and CYP11B1, respectively, might suggest a regulatory function for phosphorylated Adx in steroidogenesis. However, it has not yet been possible to identify phosphorylated Adx unequivocally in cell culture experiments or directly in bovine tissue. It cannot be excluded that the phosphorylation of Adx at position T71 might only take place in certain developmental stages. For example, it has been suggested that phosphorylation of the microsomal cytochrome CYP17, which is involved in the synthesis of androgen sex steroids, is developmentally regulated.78
Considering this, an enhancement of the CYP11A1-catalyzed formation of pregnenolone might be required. Since CYP11A1 competes with CYP11B1 for the common electron donor, a phosphorylation of Adx at residue Thr-71 could enhance the interaction with CYP11A1 and result in an increased production of the steroid precursor. Thus, phosphorylation might provide a fine-tuning mechanism that favors the formation of pregnenolone and the general steroid biosynthesis. Further in vivo experiments (e.g., primary cell culture experiments) may help to clarify the role of CK2 phosphorylation of Adx in steroidogenesis.