Before crystallization, the purified CARDO-OJ3 was confirmed to retain its angular dioxygenation activity for carbazole when the electron-transfer proteins CARDO-FCA10 and CARDO-RCA10 were supplied (data not shown). This result clearly indicates that CARDO-OJ3 can receive electrons from CARDO-FCA10.
To ensure that the two types of crystal consisted of both CARDO-OJ3 and CARDO-FCA10, SDS–PAGE followed by Western blot analysis was performed using the dissolved crystals. The SDS–PAGE analysis clearly indicated that both types of crystals contained CarAaJ3 (CARDO-OJ3 monomer; Figs. 3
a and 3
b, lanes 2 and 3). This result was further confirmed by Western blot analysis, which showed clear detection of CarAaJ3 with ~44 kDa molecular weight (data not shown). On the other hand, because the degradation peptide that originated from CarAaJ3 had a molecular size similar to that of CarAcCA10 (Fig. 3
a, lanes 1, 2 and 3), we could not conclude that both types of crystals contained CarAcCA10 (CARDO-FCA10). Western blots using anti-CarAcCA10 antibody indicated that both crystals contained CarAcCA10 protein, although the bands at about 30 kDa reacted slightly with anti-CarAcCA10 antibody (Fig. 3
Figure 3 SDS–PAGE and Western blot analysis of the dissolved crystals. (a) SDS–PAGE of the dissolved crystals (5 µg each). Lane M, low-molecular-weight markers; lane 1, dissolved crystals of CARDO-OJ3; lane 2, dissolved type I crystals (more ...)
The merged data sets for the type I and II crystals were collected to 1.90 and 2.05 Å resolution, respectively. The data-collection and processing statistics are summarized in Table 1. The space groups of both crystals were determined to be P
, with similar unit-cell parameters. During purification using gel-filtration chromatography, the molecular weights of CARDO-OJ3
were calculated to be 132 and 13 kDa (data not shown), suggesting that CARDO-OJ3
homotrimeric and monomeric configurations, respectively. These quaternary structures suggested by biochemical experiments are in accordance with the crystal structures of CARDO-OJ3
as single proteins (Nam et al.
; Nojiri et al.
). The trimeric structure of CARDO-OJ3
has three completely separate active sites (catalytic non-haem irons), suggesting that three regions capable of interacting with CARDO-FCA10
exist around three Rieske [2Fe–2S] clusters in CARDO-OJ3
. Therefore, assuming one molecule of CARDO-OJ3
and three molecules of CARDO-FCA10
per asymmetric unit, the Matthews coefficient V
) is 2.63 Å3
(corresponding to a solvent content of 53.2%). Consequently, the asymmetric unit in the crystal may contain one complex that is a trimer of a heterodimer consisting of CARDO-OJ3
Crystal parameters and data-collection statistics
We are now attempting to determine the structure of this complex by the molecular-replacement method using the determined structures of CARDO-OJ3
and the refinement is now in progress. Until now, various types of electron transport between proteins have been reported, but there are only a few reports of their three-dimensional structures. Three structures of complexes of ferredoxins with their reductases have been determined: the complexes of Anabaena
and maize ferredoxin with their respective ferredoxin:NADP+
oxidoreductase in oxygenic photosynthesis (Morales et al.
; Kurisu et al.
) and the complex of mitochondria adrenodoxin with adrenodoxin reductase in steroid biosynthesis (Müller et al.
). To the best of our knowledge, the complex structure of the terminal oxygenase with its electron-donor protein has not been determined yet, except for the complex structure of the haem- and FMN-binding domains of the bacterial cytochrome P450BM-3, a prototype for the complex between eukaryotic microsomal P450 monooxygenase and P450 reductase (Sevrioukova et al.
). Therefore, the complex structure of CARDO-OJ3
will represent the first structure of a biologically relevant complex between an oxygenase and its electron donor, ferredoxin.
Several reports have indicated that while the reductase components of ROSs can be replaced by unrelated reductases, the ferredoxin components cannot be replaced (Subramanian et al.
; Haiger & Gibson, 1990
; Fukuda et al.
). In the CARDOCA10
can be replaced by an unrelated reductase, such as the ferredoxin reductase from spinach, but CARDO-FCA10
is indispensable for electron transfer to CARDO-OCA10
, suggesting that there is a specific interaction between CARDO-OCA10
(Nam et al.
). As no functional difference has been observed between CARDO-OJ3
in several experiments, the forthcoming structure of the CARDO-OJ3
complex will provide insight into the structural basis of the specific interaction between the CARDO components. In addition, as the first structural example of an ROS electron-transport complex structure, it will also provide useful information for understanding the general architecture of the component interactions and electron transport in this important class of multicomponent oxygenase systems.