Shingo Nagano

Crystal structure of the cytochrome p450cam mutant that exhibits the same spectral perturbations induced by putidaredoxin binding.

The cytochrome P450cam active site is known to be perturbed by binding to its redox partner, putidaredoxin (Pdx). Pdx binding also enhances the camphor monooxygenation reaction (Nagano, S., Shimada, H., Tarumi, A., Hishiki, T., Kimata-Ariga, Y., Egawa, T., Suematsu, M., Park, S.-Y., Adachi, S., Shiro, Y., and Ishimura, Y. (2003) Biochemistry 42, 14507–14514). These effects are unique to Pdx because nonphysiological electron donors are unable to support camphor monooxygenation. The accompanying 1H NMR paper (Tosha, T., Yoshioka, S., Ishimori, K., and Morishima, I. (2004) J. Biol. Chem. 279, 42836–42843) shows that the conformation of active site residues, Thr-252 and Cys-357, and the substrate in the ferrous (Fe(II)) CO complex of the L358P mutant mimics that of the wild-type enzyme complexed to Pdx. To explore how these changes are transmitted from the Pdx-binding site to the active site, we have solved the crystal structures of the ferrous and ferrous-CO complex of wild-type and the L358P mutant. Comparison of these structures shows that the L358P mutation results in the movement of Arg-112, a residue known to be important for putidaredoxin binding, toward the heme. This change could optimize the Pdx-binding site leading to a higher affinity for Pdx. The mutation also pushes the heme toward the substrate and ligand binding pocket, which relocates the substrate to a position favorable for regio-selective hydroxylation. The camphor is held more firmly in place as indicated by a lower average temperature factor. Residues involved in the catalytically important proton shuttle system in the I helix are also altered by the mutation. Such conformational alterations and the enhanced reactivity of the mutant oxy complex with non-physiological electron donors suggest that Pdx binding optimizes the distal pocket for monooxygenation of camphor.

Putidaredoxin-cytochrome P450cam interaction.

Cytochrome P450cam (P450cam) catalyzes the monooxygenation of D-camphor. During the enzymatic reaction, oxyferrous, D-camphor-bound P450cam forms a binary complex with reduced putidaredoxin as an obligatory reaction intermediate. We have found that reduced putidaredoxin undergoes EPR-detectable conformational changes upon formation of the intermediate complex and also upon formation of a binary complex with CO- or NO-ferrous, D-camphor-bound P450cam. The structural changes in putidaredoxin are almost identical irrespective of the ligand bound to P450cam, and distinct from and significantly larger than those induced by unliganded ferrous P450cam. The binary complex formation also induce conformational alterations in the CO- and NO-ferrous, D-camphor-bound P450cam, thereby evoking simultaneous changes in the structure of the two proteins. A molecular basis and roles of such structural changes in the D-camphor monooxygenation are discussed.

Putidaredoxin-cytochrome p450cam interaction. Spin state of the heme iron modulates putidaredoxin structure.

During the monooxygenase reaction catalyzed by cytochrome P450cam (P450cam), a ternary complex of P450cam, reduced putidaredoxin, andd-camphor is formed as an obligatory reaction intermediate. When ligands such as CO, NO, and O2 bind to the heme iron of P450cam in the intermediate complex, the EPR spectrum of reduced putidaredoxin with a characteristic signal at 346 millitesla at 77 K changed into a spectrum having a new signal at 348 millitesla. The experiment with O2 was carried out by employing a mutant P450cam with Asp251 → Asn or Gly where the rate of electron transfer from putidaredoxin to oxyferrous P450cam is considerably reduced. Such a ligand-induced EPR spectral change of putidaredoxin was also shown in situ inPseudomonas putida. Mutations introduced into the neighborhood of the iron-sulfur cluster of putidaredoxin revealed that a Ser44 → Gly mutation mimicked the ligand-induced spectral change of putidaredoxin. Arg109 and Arg112, which are in the putative putidaredoxin binding site of P450cam, were essential for the spectral changes of putidaredoxin in the complex. These results indicate that a change in the P450cam active site that is the consequence of an altered spin state is transmitted to putidaredoxin within the ternary complex and produces a conformational change of the 2Fe–2S active center.

Subunit structure of recombinant rat liver l-tryptophan 2,3-dioxygenase

Abstract Rat liver l -tryptophan 2,3-dioxygenase (TDO) with a histidine tag at the N-terminus was expressed in Escherichia coli JM109 harboring plasmid pUC18 carrying the full-length cDNA of TDO. The recombinant enzyme was purified to near homogeneity by employing conventional purification methods including nickel-chelate immobilized resin column chromatography. The purified enzyme had a turnover number per heme 303 min −1 with similar spectral properties to those of native rat liver enzyme. SDS-PAGE of purified TDO preparation showed two distinct bands with molecular masses of 49 and 46 kDa. N-terminal sequence analysis of the components revealed that the 46-kDa species is shorter than the 49-kDa one by 19 amino acid residues including six histidine residues at the end. Thus, a limited proteolysis appeared to occur between Tyr 13 and Thr 14 of the original polypeptide chain. The construct of recombinant TDO with deletion of the N-terminal 13 residues gave a single band on SDS-PAGE with a molecular mass of about 46 kDa. The N-terminal truncation had no effect on the catalytic activity nor on the spectral properties.

Molecular Mechanism of Metal-Containing Oxygenases

Over a past few years, we have determined crystal structures of several metal-containing oxygenases including indoleamin-2, 3-dioxygenase and some P450s which have interesting chemistry and play important roles in metabolism. Molecular mechanisms of these oxygenases are discussed based on the crystal structures.