Tyr-143 facilitates interdomain electron transfer in flavocytochrome b2

Abstract

The role of Tyr-143 in the catalytic cycle of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) has been examined by replacement of this residue with phenylalanine. The electron-transfer steps in wild-type and mutant flavocytochromes b2 have been investigated by using steady-state and stopped-flow kinetic methods. The most significant effect of the Tyr-143----Phe mutation is a change in the rate-determining step in the reduction of the enzyme. For wild-type enzyme the main rate-determining step is proton abstraction at the C-2 position of lactate, as shown by the 2H kinetic-isotope effect. However, for the mutant enzyme it is clear that the slowest step is interdomain electron transfer between the FMN and haem prosthetic groups. In fact, the rate of haem reduction by lactate, as determined by the stopped-flow method, is decreased by more than 20-fold, from 445 +/- 50 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 21 +/- 2 s-1 in the mutant enzyme. Decreases in kinetic-isotope effects seen with [2-2H]lactate for mutant enzyme compared with wild-type, both for flavin reduction (from 8.1 +/- 1.4 to 4.3 +/- 0.8) and for haem reduction (from 6.3 +/- 1.2 to 1.6 +/- 0.5) also provide support for a change in the nature of the rate-determining step. Other kinetic parameters determined by stopped-flow methods and with two external electron acceptors (cytochrome c and ferricyanide) under steady-state conditions are all consistent with this mutation having a dramatic effect on interdomain electron transfer. We conclude that Tyr-143, an active-site residue which lies between the flavodehydrogenase and cytochrome domains of flavocytochrome b2, plays a key role in facilitating electron transfer between FMN and haem groups.

Roles of key active-site residues in flavocytochrome P450 BM3

“…For example, the tetrameric -lactate cytochrome c oxidoreductase (flavocytochrome b # ) from Saccharomyces cere isiae uses Arg-376 and Tyr-143 to stabilize the binding of lactate. The flavocytochrome b # mutant Y143F shows a 6-fold increased K m and an unchanged k cat relative to wild-type using -lactate as a substrate and ferricyanide as the electron acceptor [39,40]. The motif is also conserved in flavocytochrome b # from Hansenula anomala (Arg-362 and Tyr-135) [41] and in the closely related Rhodotorula graminis -mandelate dehydrogenase (Tyr-25 and Arg-277), which is also a tetrameric flavocytochrome [42].…”

Structural Analysis of Respirasomes in Electron Transfer Pathway of Acidithiobacillus ferrooxidans: A Computer-Aided Molecular Designing Study

“…Further, the presence of Phe312 of Cyc2 at intermediate position between heme C and Asp73 could be presumed an important factor influencing electron transfer from the donor site of heme C to the acceptor site of Asp73 (Figure 2(b)). Previous studies on the cytochromes had clearly demonstrated the significance of phenylalanine/tyrosine residues in mediating electron transfer [58,59]. A close visual inspection of the core region of RcY structure after MD simulation allowed us to observe that several hydrophobic interactions comprising the residues Phe125, Val74, Phe76, Phe54, Phe87, and Trp7 form a channel required for electron transfer to the Cu center (Figure 4(a)).…”

On the lack of coordination between protein folding and flavin insertion in Escherichia coli for flavocytochrome b2 mutant forms Y254L and D282N

“…9). This helix is normally rather close to the flavin because Y143 is an active-site residue that forms a hydrogen bond with the substrate carboxylate Miles et al, 1992;Rouviere-Fourmy et al, 1994). Moreover, the two terminal nitrogens in the side chain of R376, another active-site residue, form good hydrogen bonds to the peptide carbonyl group of Y143 and may also interact with the carbonyl oxygen atoms of Y 144 and/or S146.…”

“…Y254F mutant proteins, in particular, were studied in detail (Dubois et al, 1990;Miles et al, 1992;Rouvikre-Fourmy et al, 1994). Although their physical properties were not specifically characterized, these mutant enzymes showed no manifest difference with the wild-type protein, either during production in E. coli or throughout purification, storage, and handling.…”