FMN semiquinone

Although the measured E pg for FMN is higher than the potential of the substrate-bound heme, reduction of the heme hy the FMN semiquinone is observed experimentally, suggesting that some shift of relative midpoint potentials occurs during turnover. 

Laser flash photolysis studies using the deazariboflavin system [80, 81] have shown that pyruvate binding also exerts a strong influence on intramolecular ET. In the one-electron reduced enzyme, ET from the FMN semiquinone to the oxidized heme can be observed only in the presence of pyruvate for this reaction, k j = 500 s. When the enzyme is completely reduced by stoichiometric addition of lactate prior to laser photolysis with dRf alone, pyruvate binding inhibits ET from fully reduced flavin to oxidized heme. This reaction has an observed /cet = 2000 s in the absence of pyruvate [81]. Similar values for these two rate constants have been obtained by temperature-jump measurements [82]. 

Electron flow within the protein proceeds from the reducing substrate (NADPH) to FAD to FMN to heme. The initial reduction forms fully reduced FAD (FADH2), which then transfers one-electron to FMN forming the neutral FAD semiquinone (FADH ) and the anionic FMN semiquinone (FMN ). When the fatty acid substrate is bound within a long hydrophobic channel adjacent to the distal face of the heme, it induces a low-spin to high-spin change and an increase in the heme potential [93]. This probably involves displacement of the water molecule in the sixth coordination position [100] and a decrease in the local dielectric con-... 

Several residues involved in interactions with the FMN have been identified (25,39). Of particular note is the interaction of Lys 349 with N1 and 02 of the isoalloxazine ring. Such interactions have been suggested to stabilize the anionic form of the FMN semiquinone and hy-droquinone and to account for the enhanced reactivity of flavin N5... 

Mechanism of cytochrome P450 reductase from the house fly Evidence for an FMN semiquinone as electron donor. FEBS Lett. 453, 201-204. 

Figure 2.4 illustrates the overall reaction mechanism by which two-electrons from NADFH are transferred to the one-electron acceptor, ferric F450. Two electrons from NADFH must enter the enzyme as a hydride ion to the FAD, followed by intramolecular electron transfer to FMN. The FMN semiquinone is extremely stable, indicating that it is the hydroquinone FMN that transfers electrons to electron acceptors and that the fully oxidized enzyme form does not accumulate. The FOR flavins cycle in a 1-3-2-1 electron cycle (upper half circle in Fig. 2.4a). The air-stable form, FMN /FAD can be formed from the fully oxidized form during the priming reaction (Fig. 2.4b). At high concentrations of NADFH, the intermediate FMNH2/FAD is reduced to a four-electron reduced form [33, 34], Since the air-stable semiquinone form is found predominantly in hver microsomes [26], the 1-3-2-1 cycle is likely the major mechanism in vivo. Although the low reduction potential of FAD, near...

On binding to the apoflavodoxin the midpoint redox potentials of the FMN are drastically altered and the FMN semiquinone becomes much more stable, thus allowing flavodoxin to function as a one-electron transfer centre in which the flavin cycles between the semiquinone and fully reduced oxidation states. An interesting aspect of cofactor binding is that in most flavodoxins the isoalloxazine ring is sandwiched between two aromatic residues one is a highly conserved tyrosine (Y94) and the other usually a tryptophan (W57). 

Murataliev and Feyereisen have investigated the interaction of recombinant Musca domestica (house fly) P450 reductase with NADPH and the role of the FMN semiquinone in reducing cytochrome The kinetics of the latter process together with EPR spectroscopy revealed that the enzyme has two types of neutral FMN radical. One serves as the catalytic intermediate of cytochrome c reduction while the other is air-stable, reducing cytochrome c 3000 times more slowly. 

The second step involves the transfer of electrons from the reduced [FMNHg] to a series of Fe-S proteins, including both 2Fe-2S and 4Fe-4S clusters (see Figures 20.8 and 20.16). The unique redox properties of the flavin group of FMN are probably important here. NADH is a two-electron donor, whereas the Fe-S proteins are one-electron transfer agents. The flavin of FMN has three redox states—the oxidized, semiquinone, and reduced states. It can act as either a one-electron or a two-electron transfer agent and may serve as a critical link between NADH and the Fe-S proteins. 

Component B is a monomeric reductase with a molecular weight of 35,000 and contains per mol of enzyme, 1 mol of FMN, 2.1 mol of Fe, and 1.7 mol of labile sulfur. After reduction with NADH, the ESR spectrum showed signals that were attributed to a [2Fe-2S] structure and a flavo-semiquinone radical (Schweizer et al. 1987). The molecular and kinetic properties of the enzyme are broadly similar to the Class IB reductases of benzoate 1,2-dioxygenase and 4-methoxybenzoate monooxygenase-O-demethylase. 

Flavin redox states in a dual flavin enzyme. (Left) Single-electron reduction of the isoalloxazine ring generates the semiquinone radical, while reduction by two electrons generates the fully reduced species. (Right) Five possible oxidation levels of a dual flavin enzyme, where the FMN reduction potential is held at a more positive value relative U) FAD. The flavins can theoretically accept a maximum of four electrons obtained from two NADPH. However, in NADPH-cytochrome P450, reductase, full reduction of the flavins is not normally reached when NADPH serves as the reductant. 

All known flavodoxins have low molecular weights (14,500-23,000 g mole" ) and contain a single polypeptide chain and a single bound FMN. Upon the addition of one reducing equivalent, the FMN adds one electron and one proton to form the neutral semiquinone. This species is blue in color (as contrasted to the yellow color of the oxidized form) and has broad absorption bands in the visible between 400 and 700 nm. Addition of a second equivalent leads to a one-electron reduction to the hydroquinone, which is pale yellow in color and has weak bands at around 450 and 365 nm. 

A comprehensive series of oxidation-reduction potential measurements have shown the FAD moiety to have the following one-electron couples PFl/PFIH = = —290 mV and PFIH 7PFIH2 = —365 mV while the FMN moiety exhibits the following PFl/PFl- = -110 mV and PFIH /PFIH = -270 mV. The FMN and FAD smiquinones were found to both be the neutral form as judged from absorption and ESR spectral data. The overlap of oxidized/semiquinone potential of the FAD moiety withkhe semiquinone/hydroquinone couple of the FMN moiety demonstrates the thermodynamic facilitation of flavin-flavin electron transfer via a one-electron mechanism. Stopped-flow kinetic data are also consistent with this view in... 

FMN consists of the structure above the dashed line on the FAD (oxidized form). The flavin nucleotides accept two hydrogen atoms (two electrons and two protons), both of which appear in the flavin ring system. When FAD or FMN accepts only one hydrogen atom, the semiquinone, a stable free radical, forms... 

The functional end of the flavin coenzymes FMN and FAD is the tricyclic isoalloxazine system, with the numbering system shown in structure I, the air-stable, yellow, oxidized form. The other two functionally important redox states are the one-electron-reduced semiquinone, II (pKa = 8.4 for dissociation at N(5)), and the two-electron-reduced, colorless dihydroflavin, III. In the dihydro form N(5), C(4a), C(la), andN(l) form a diaminoethylene system and it was anticipated that nitrogen at the 5 and 1 positions would be key to coenzymatic function. 

During catalytic turnover, NADPH reduces FAD and the FAD subsequently reduces FMN by two electrons with the latter eyeling between the hydroquinone and semiquinone states while carrying out the 1-electron reduction of the P450 heme iron (Masters et al., 1966 Backes and Reker-Backes, 1988). The midpoint redox potentials of the FAD are El(,x/sq n290 mV and fi365 mV and of the FMN are El(,x/sq nl lOmV and... 

The electron transfer rates in P450BM3, measured by laser flash photolysis using semicarbazide-aetivated 5-deazflavin semiquinone, show that no reduction of the native BMP heme oeeurs even though FMN could be reduced rapidly to the semiquinone (Hazard et al., 1997). In the presence of earbon monoxide, which can displace water from the sixth coordination site of iron and convert the low-spin ferric iron to high spin, the intramolecular electron transfer rate is 18sec". In the presence of both CO and the substrate myristic acid, an intramolecular electron transfer rate of up to 250sec" can be obtained. 

---