Protein conformational gating of enzymatic activity in xanthine oxidoreductase

Abstract

In mammals, xanthine oxidoreductase can exist as xanthine dehydrogenase (XDH) and xanthine oxidase (XO). The two enzymes possess common redox active cofactors, which form an electron transfer (ET) pathway terminated by a flavin cofactor. In spite of identical protein primary structures, the redox potential difference between XDH and XO for the flavin semiquinone/hydroquinone pair (E(sq/hq)) is ~170 mV, a striking difference. The former greatly prefers NAD(+) as ultimate substrate for ET from the iron-sulfur cluster FeS-II via flavin while the latter only accepts dioxygen. In XDH (without NAD(+)), however, the redox potential of the electron donor FeS-II is 180 mV higher than that for the acceptor flavin, yielding an energetically uphill ET. On the basis of new 1.65, 2.3, 1.9, and 2.2 Å resolution crystal structures for XDH, XO, the NAD(+)- and NADH-complexed XDH, E(sq/hq) were calculated to better understand how the enzyme activates an ET from FeS-II to flavin. The majority of the E(sq/hq) difference between XDH and XO originates from a conformational change in the loop at positions 423-433 near the flavin binding site, causing the differences in stability of the semiquinone state. There was no large conformational change observed in response to NAD(+) binding at XDH. Instead, the positive charge of the NAD(+) ring, deprotonation of Asp429, and capping of the bulk surface of the flavin by the NAD(+) molecule all contribute to altering E(sq/hq) upon NAD(+) binding to XDH.

Figure 1

Cofactor arrangements in xanthine oxidoreductase. The 423-433 loops in the neighborhood of the flavin binding sites are depicted as pink (XDH) and green (XO) threads, respectively.

Figure 2

Structural overview of the location of the flavin ring of FAD and the iron-sulfur clusters (FeS-I and FeS-II) in XDH (left) and XO (right). The 423-433 loops in the neighborhood of the flavin binding sites are depicted as pink (XDH) and green (XO) threads, respectively.

Figure 3

(a) Redox titration for the flavin E_sq/hq value (■) versus solvent potential (E_solv) in XDH and XO. Associated changes in the protonation state of Asp429 as deprotonation state (i.e., 1.0 for fully deprotonated ionized state and 0.0 for fully protonated neutral state) are also shown (○). (b) Shift in the titration curves upon NAD⁺ binding (solid black arrow). The NAD⁺ redox state is fixed while titrating E_sq/hq. For definitions of protonation and redox probabilities, see Ref. .

Figure 3

(a) Redox titration for the flavin E_sq/hq value (■) versus solvent potential (E_solv) in XDH and XO. Associated changes in the protonation state of Asp429 as deprotonation state (i.e., 1.0 for fully deprotonated ionized state and 0.0 for fully protonated neutral state) are also shown (○). (b) Shift in the titration curves upon NAD⁺ binding (solid black arrow). The NAD⁺ redox state is fixed while titrating E_sq/hq. For definitions of protonation and redox probabilities, see Ref. .

Figure 4

Protein environment of the FAD binding site of XDH (left) and XO (right) and the H-bond network (bottom). N and O atoms are depicted as blue and red balls, respectively. Possible H-bonds, in particular those among Glu263, Glu267, Arg394, and Asp430 are indicated by pink solid lines. P_proximal stands for the proximal phosphate atom of the diphosphate region. — — and // // indicate the presence and absence of an H-bond, respectively, with the numbers giving its length in Ångstrom units.

Figure 4

Protein environment of the FAD binding site of XDH (left) and XO (right) and the H-bond network (bottom). N and O atoms are depicted as blue and red balls, respectively. Possible H-bonds, in particular those among Glu263, Glu267, Arg394, and Asp430 are indicated by pink solid lines. P_proximal stands for the proximal phosphate atom of the diphosphate region. — — and // // indicate the presence and absence of an H-bond, respectively, with the numbers giving its length in Ångstrom units.

Figure 5

Electron density maps of the FAD and NAD(H) binding sites in (a) ligand-free XDH, (b) ligand-free XO, (c) the XDH-NADH complex, and (d) the XDH-NAD⁺ complex. For clarity, only the FAD, NAD(H), and Asp429 are shown in atom representation. The backbone trace of the 423-433 loop is indicated by a green thread. Although the loop seems to swing away from the flavin ring in XO it does interfere with dinucleotide access in its new location.

Figure 5

Electron density maps of the FAD and NAD(H) binding sites in (a) ligand-free XDH, (b) ligand-free XO, (c) the XDH-NADH complex, and (d) the XDH-NAD⁺ complex. For clarity, only the FAD, NAD(H), and Asp429 are shown in atom representation. The backbone trace of the 423-433 loop is indicated by a green thread. Although the loop seems to swing away from the flavin ring in XO it does interfere with dinucleotide access in its new location.

Figure 5

Electron density maps of the FAD and NAD(H) binding sites in (a) ligand-free XDH, (b) ligand-free XO, (c) the XDH-NADH complex, and (d) the XDH-NAD⁺ complex. For clarity, only the FAD, NAD(H), and Asp429 are shown in atom representation. The backbone trace of the 423-433 loop is indicated by a green thread. Although the loop seems to swing away from the flavin ring in XO it does interfere with dinucleotide access in its new location.

Figure 5

Electron density maps of the FAD and NAD(H) binding sites in (a) ligand-free XDH, (b) ligand-free XO, (c) the XDH-NADH complex, and (d) the XDH-NAD⁺ complex. For clarity, only the FAD, NAD(H), and Asp429 are shown in atom representation. The backbone trace of the 423-433 loop is indicated by a green thread. Although the loop seems to swing away from the flavin ring in XO it does interfere with dinucleotide access in its new location.

Figure 6

Shift in the redox potential levels and energetics for ET from FeS-II to flavin (sq/hq) in response to NAD⁺ binding. The solid black arrows indicate the redox potential shifts due to the NAD⁺ binding.

Figure 7

Representation of the FAD site of XDH. In the left hand figure, the NADH molecule has been removed to indicate the water-accessible area of the flavin ring. In the right hand figure, the same view is given but with the NADH molecule included to show how well the NADH molecule covers the flavin ring. The FAD atoms are depicted as ball-and-stick while the NADH molecule (cyan) and the protein atoms (yellow) are depicted as space-filling models.