Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

5A, 6A, 18B, NW12/2003G115

Crystallographic analysis of complex structures of fungal denitrifying enzyme
P450nor
Rieko OSHIMA1, Shinya FUSHINOBU1*, Fei SU2, Li ZHANG2, Naoki TAKAYA2,Hirofumi SHOUN1
1
Dept. of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
2
Institute of Applied Biochemistry, Univ. of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.
Introduction
P450nor (CYP55) functions as nitric-oxide reductase toreduce NO to N2O, in the denitrification system of afungus, Fusarium oxysporum [1].
++2NO + NAD(P)H + H → N2O + NAD(P) + H2OP450nor can perform this reaction without the aid ofother redox partners, directly transferring two electrons
from NAD(P)H to bound heme [2]. The reaction isunusual as compared with other monooxygenase
cytochrome P450s. Electron transfer from NAD(P)H toan one-electron redox center usually requires the aid ofother redox partner(s) such as a flavoprotein. Therefore,
the electron transfer catalyzed by P450nor apparently
contradicts the common sense of biological electrontransfer. Moreover, P450nor is shown to catalyze N2Oforming "codenitrification" reaction [3].
To elucidate the reaction mechanism of this unusual
P450 enzyme, we tried to obtain complex structures withthe dinucleotide-cofactor or its analogues.
Manuscript preparation
We succeeded in crystallizing a complex form ofP450nor with an NADH analogue, nicotinic acid pyridine
dinucleotide (NAAD), utilizing a double mutant(Ser73Gly/Ser75Gly) of the enzyme [4], whose affinityfor NAD(P)H and its analogues was remarkably
improved. The dataset up to 1.8 Å resolution wascollected at BL6A, and the crystal structure was refined
to R = 20.6% and Rfree = 24.3% [5] (Fig. 1). The complex
structure provided conclusive evidence for themechanism of the unprecedented electron transfer.
Comparison of the structure with those of dinucleotidefree forms [6] revealed a global conformational change,
not a hinge motion, accompanied by local movements
caused by the binding of the pyridine nucleotide into theheme-distal pocket. Arg64 and Arg174 [7] fix thepyrophosphate moiety upon the dinucleotide-binding.
Most of the charged residues in the pocket are clusteredon the 'Arg64-side', whereas Arg174 is the sole charged
residue on the opposite side. A salt bridge network(Glu71-Arg64-Asp88) at the 'Arg64-side' is shown to be
crucial for a high catalytic turnover of this enzyme [8, 9].
Electron donor specificity for NADH and NADPH ismaintained by the size of the side chains of the residues in
the B'-helix, which is located on the top of the 'Arg64side'. Stereo-selective hydride transfer from NADH toNO-bound heme [7] was strongly suggested from thestructure, the nicotinic acid ring being fixed near theheme by the conserved Thr residue in the I-helix and theupward-shifted propionate side chain of the heme. Aproton channel near the NADH channel is formed upon
the dinucleotide-binding, which should direct continuoustransfer of the hydride and proton.

Figure 1 Ribbon diagram of the crystal structure ofP450nor complexed with NAAD.

Reference[1] K. Nakahara, et al., J. Biol. Chem. 268, 8350 (1993).
[2] Y. Shiro, et al., J. Biol. Chem. 270, 1617 (1995).
[3] F. Su et al., Biosci. Biotechnol. Biochem. 68, 473(2004)[4] L. Zhang, et al., J. Biol. Chem. 277, 33842 (2002).
[5] R. Oshima, et al., J. Mol. Biol. 342, 207 (2004).
[6] H. Shimizu et al, J. Biol. Chem. 275, 4816 (2000).
[7] T. Kudo et al, J. Biol. Chem. 276, 5020 (2001).
[8] M. Umemura et al., Eur. J. Biochem. 271, 2887(2004)[9] F. Su et al., Biosci. Biotechnol. Biochem. 68, 1156(2004)[10] T. Daiber et al, J. Inorg. Biochem. 88, 343 (2002).
* asfushi@mail.ecc.u-tokyo.ac.jp

Users' Report239