Effects of Flavin-Binding Motif Amino Acid Mutations in the NADH-Cytochrome b5 Reductase Catalytic Domain on Protein Stability and Catalysis

Abstract

Porcine NADH-cytochrome b5 reductase catalytic domain (Pb5R) has the RXY(T/S)+(T/S) flavin-binding motif that is highly conserved among the structurally related family of flavoprotein reductases. Mutations were introduced that alter the Arg(63), Tyr(65), and Ser(99) residues within this motif. The mutation of Tyr(65) to either alanine or phenylalanine destabilized the protein, produced an accelerated release of FAD in the presence of 1.5 M guanidine hydrochloride, and decreased the k(cat) values of the enzyme. These results indicate that Tyr(65) contributes to the stability of the protein and is important in the electron transfer from NADH to FAD. The mutation of Ser(99) to either alanine or valine, and of Arg(63) to either alanine or glutamine increased both the K(m) values for NADH (K(m)(NADH)) and the dissociation constant for NAD(+) (K(d)(NAD+)). However, the mutation of Ser(99) to threonine and of Arg(63) to lysine had very little effect on the K(m)(NADH) and K(d)(NAD+) values, and resulted in small changes in the absorption and circular dichroism spectra. These results suggest that the hydroxyl group of Ser(99) and the positive charge of Arg(63) contribute to the maintenance of the properties of FAD and to the effective binding of Pb5R to both NADH and NAD(+). In addition, the mutation of Arg(63) to either alanine or glutamine increased the apparent K(m) values for porcine cytochrome b5 (Pb5), while changing Arg(63) to lysine did not. The positive charge of Arg(63) may regulate the electron transfer through the electrostatic interaction with Pb5. These results substantiate the important role of the flavin-binding motif in Pb5R.

Role of Thr66 in Porcine NADH-cytochromeb 5 Reductase in Catalysis and Control of the Rate-limiting Step in Electron Transfer

“…It seems that the apparent K m NADH value of Pb5R is mainly dependent on the affinity for 5Ј-ADP or the 5Ј-ADP-ribose moiety of the pyridine nucleotide. We have previously reported that the R63A and R63Q mutants of Pb5R did not bind to 5Ј-ADP-agarose and suggested that Arg 63 in Pb5R assists the binding of NAD ϩ and NADH by reducing electrostatic repulsion between the negative charges on the phosphates of pyridine nucleotide and FAD (28). In addition, replacement of the Thr 153 and Thr 156 residues in Pb5R, which are positioned close to the potential binding site of the nicotinamide moiety of NADH (Fig.…”

“…In the Pb5R, Arg 63 , Tyr 65 , and Thr 66 comprise this sequence motif (19). Using sitedirected mutagenesis, we demonstrated that the positive charge of Arg 63 is critical for the affinities of Pb5R for both NADH and NAD ϩ , and the specific arrangement between the side chain of Tyr 65 and FAD contributes to protein stability and electron transfer (28). Marohnic and Barber (29) also reported the effects of mutations of the corresponding Arg 91 in rat b5R.…”

“…Measurement of Dissociation Constants-The dissociation constant of the oxidized enzyme for NAD ϩ (K d NADϩ ) was determined by measuring the perturbation of the flavin spectrum as previously described (28). Approximately 40 M of oxidized mutant proteins were titrated with NAD ϩ in 10 mM potassium phosphate (pH 7.0) at 25°C.…”

“…The Thr 66 residue in Pb5R is positioned near both the N5 atom of the isoalloxazine ring of FAD and the potential binding site of the nicotinamide ring of NADH (20,28) (Fig. 1).…”

Molecular Dynamics Simulation Study on Stabilities and Reactivities of NADH Cytochrome B5 Reductase

“…On the other hand, using the conventional model A with the alanine scanning method, Tyr93Ala mutant becomes the favorable mutation with ∆ ∆G total A ) -3.8 kcal/mol, in which the negative change in the solvation free energy ∆∆G PB A overcomes the unfavorable electrostatic and VDW interactions, ∆∆E electrostatic and ∆∆E vdW , which contradicts with the experimental results. Kimura et al 17 have pointed out that Tyr93 makes an aromatic contact with the si-face of the isoalloxazine ring through a direct hydrogen bond to the 4′-hydroxyl group of the ribityl moiety of the FAD cofactor (see Figure 4a). In the case of the Tyr93Ala mutant, the weakening of the VDW interaction, ∆∆E vdW ) 6.4 kcal/ mol, indicates that the VDW contact between the phenyl ring of Tyr93 and the isoalloxazine ring of FAD in the wild-type enzyme plays an important role in the stability of the complex as suggested by Kimura et al 17 Usually, we can easily calculate the binding free energies for mutants by means of the alanine scanning approach, which uses a single trajectory obtained for a wild-type MD simulation to generate the structures of monomers and dimers.…”