FAD domain

Figure 17.3 The active site structure of xanthine oxidoreductase and the structure of the bovine enzyme with the two Fe-S domains (green and blue), the FAD domain (grey) and the molybdenumbinding domain (red). (From Hille, 2005. Copyright 2005, with permission from Elsevier.)...

Thus for dehydrogenase partial activities of NR, the 28kDa FAD domain alone is a NADH ferricyanide reductase (NADH FR) and the FAD domain linked to the haem domain (40kDa) is a NA DH cytochrome c reductase (NADH CR) as well as a NADH dichloro-phenolindophenol reductase (NADH.DR). As for the nitrate-reducing activities, the methylviologen nitrate reductase (MV NR) and the reduced flavin nitrate reductase (FMN NR) activities involve the MoCo domain (75 kDa) linked to the haem domain, whereas reduced brom-... 

Group-specific modifications of Cb5R have shown similarly that a tyrosine participates in FAD binding (Strittmatter, 1961). Two tyrosines are conserved (Tyr719 and Tyr757 in tobacco) both of which lie in the N-terminal half of the FAD domain. 

Yubisui etal., 1991) and this knowledge will be of great help in obtaining further insights into the structure-function relationships of NR. A similar 3-dimensional structure is expected, because of the mean 50% sequence homology between Cb5R and the NR FAD domain. 

The crystal structures of Ffr from two Shewanella spp. and Ifc have been determined and are very similar to each other. The FAD domain of these flavocytochromes has significant structnral similarity to other FAD-binding proteins. The heme-binding domain shows very tittle secondary structure. All of the hemes are coordinated by two histidines and are in close distance to each other. Hemes 1 and 2 are positioned in a perpendicular motif, whereas hemes 2 and 3 are in a parallel stacked motif These three hemes can be superimposed to hemes 5 7 of HAO and 2 4 of NrfA (Figure 3). Heme 4 of Ffr is deviated from the corresponding heme 8 position in HAO, because it is oriented toward the FAD group. 

Mammalian XO is a homodimer of around 1330 amino acids which binds a number of electron transfer centres — an FAD, two spectroscopically distinct [2Fe-2S] clusters, and the Mo-cofactor. The structure of the bovine xanthine dehydrogenase (XDH), bound to the competitive inhibitor salicylic acid, and presented in Figure 17.3(a), consists of four domains, two Fe/S domains (1 and 11) in the N-terminal portion of the molecule, followed by the central FAD domain and the molybdenum-binding domain in the C-terminal part of the molecule. 

Color Plate 13. Ribbon diagram of the pea ferredoxin-NADP reductase (Tyr308->Ser)-NADP complex. The N-terminal FAD domain and the C-terminal NADP domain are coiored in teai and red, respectively FAD in yellow and NADP in green. (Courtesy of Dr. P. A. Karplus). [See Chapter 34, Fig. 21.]... 

Figure 3.12. The crystal structure of P450 reductase. Note that the FMN and FAD are in direct contact. The schematic diagram illustrates that it may be necessary for the FMN and FAD domains to separate to enable the FMN domain to dock onto the P450 prior to the FMN-to-heme electron transfer reaction.

Figure 4.9. Kinetic schemes for the isolated FAD domain (upper scheme) and full-length CPR (lower scheme) derived from stopped-flow studies (taken from Gutierrez ). The reversible nature of the reaction is indicated, as is the presence of the second nicotinamide coenzyme-binding site (indicated in parenthesis for the FAD domain). For clarity, only enzyme intermediates bound with a single coenzyme are shown in the lower scheme for CPR, but reference to the upper scheme indicates that a second molecule of NADPH has the capacity to bind to the enzyme.

The presence of two coenzyme-binding sites is unexpected since they cannot be inferred solely from the crystal structure of CPR. Kinetic studies with wild t) e and W676H CPR at different concentrations of NADPH have, however, provided further support for the existence of two sites The rate of flavin reduction in the isolated FAD domain and CPR increases as NADPH is decreased from molar excess to stoichiometric concentrations. At stoichiometric concentration, the second noncatalytic site is predominantly vacant and the partial inhibition on the rate of flavin reduction from the catalytic site is therefore relieved (Figure 4.9). Occupation of the noncatalytic site occurs at NADPH concentrations in excess of the enzyme concentration, and impairs NADP" " release from the catalytic site. This in turn partially inhibits flavin reduction, the rate of which is gated by NADP release. Preincubation of the enzyme with a stoichiometric amount of adenosine 2, 5 -diphosphate does not lead to inhibition of the flavin reduction rate. We infer that the binding of adenosine 2, 5 -diphosphate prevents NADPH from binding to the noncatalytic site. This observation also suggests that it is the nicotinamide-ribose-phosphate portion of NADPH bound at the second site that hinders NADP" release from the catalytic site. Clearly, these new... 

It Is Interesting to examine In the crystal structure what are the functional roles of these five regions In human GR. The first region from residue 25 to residue 157 In the graph corresponds to the FAD domain from residue 18 to residue 157 In the crystal structure. The second and a half of the third regions In the graph constructs the NADPH doamln from residue 158 to residue 293 In the... 

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