Structural analysis of reaction intermediates of cytochrome reductase

Yamada, Mitsugu; Tamada, Taro; Matsumoto, Fumiko; Takeda, Kazuki*; Kimura, Shigenobu*; Kuroki, Ryota; Miki, Kunio*

NADH-cytochrome reductase (b5R; EC 1.6.2.2) is a flavoprotein and catalyses two-electron transfer from NADH to cytochrome through FAD. Here, we present crystal structures of fully reduced and two re-oxidized forms of b5Rs determined. The crystal structure of fully reduced b5R showed that the relative location of two domains slightly shifted in comparison with that of oxidized form. This shift allowed to create a new hydrogen bonding interaction between N5 atom of isoalloxazine ring and hydroxyl oxygen atom of Thr66. The isoalloxazine ring of FAD in fully reduced form is flat and stacked with the nicotinamide ring of bound NAD. Moreover, two re-oxidized forms (form-1 and 2) were prepared using fully reduced crystals by exposure to the air. The electron densities for the nicotinamide ring of NAD was ambiguous in form-1 and was completely disappeared in form-2. These results suggested that re-oxidization follows a two-step mechanism in which nicotinamide moiety is released firstly and then whole NAD is released.