Crystallographic Study of the Recombinant Flavin-Binding Domain of Baker's Yeast Flavocytochrome b₂:  Comparison with the Intact Wild-Type Enzyme

Abstract

Flavocytochrome b(2) catalyzes the oxidation of L-lactate to pyruvate and the transfer of electrons to cytochrome c. The enzyme consists of a flavin-binding domain, which includes the active site for lacate oxidation, and a b(2)-cytochrome domain, required for efficient cytochrome c reduction. To better understand the structure and function of intra- and interprotein electron transfer, we have determined the crystal structure of the independently expressed flavin-binding domain of flavocytochrome b(2) to 2.50 A resolution and compared this with the structure of the intact enzyme, redetermined at 2.30 A resolution, both structures being from crystals cooled to 100 K. Whereas there is little overall difference between these structures, we do observe significant local changes near the interface region, some of which impact on amino acid side chains, such as Arg289, that have been shown previously to have an important role in catalysis. The disordered loop region found in flavocytochrome b(2) and its close homologues remain unresolved in frozen crystals of the flavin-binding domain, implying that the presence of the b(2)-cytochrome domain is not responsible for this positional disorder. The flavin-binding domain interacts poorly with cytochrome c, but we have introduced acidic residues in the interdomain interface region with the aim of enhancing cytochrome c binding. While the mutations L199E and K201E within the flavin-binding domain resulted in unimpaired lactate dehydrogenase activity, they failed to enhance electron-transfer rates with cytochrome c. This is most likely due to the disordered loop region obscuring all or part of the surface having the potential for productive interaction with cytochrome c.

High Resolution Structures of an Oxidized and Reduced Flavoprotein

“…3). In FCB2, there is a 7-residue segment near the center of the sequence that is disordered (26), and in GOX a 9-residue segment, also central to the sequence, is disordered (25). Both of these segments are located between strand ␤-3 and helix ␣-3 of the TIM barrel and are flanked in sequence by several active site residues.…”

QM/MM study ofl-lactate oxidation by flavocytochrome b2

“…1 Each of the four subunits contains a Flavin MonoNucleotide visualization are treated by VMD. 4 Several Fcb2 crystal structures 3,5 show pyruvate, the reaction product, bound in the active site on the flavin si-side, and its interactions with a number of residues. These are displayed in Figure 1.…”

“…R289 requires particular attention as it has been found in two possible conformations as depicted in Figure 2. In a few crystal structures containing pyruvate, 3,5 R289 is found in a so-called distal orientation and interacts both with D292 and a water molecule.…”

“…3,6 In the crystal structure of the R289K variant in complex with sulfite, the water molecule has been displaced by the lysine side chain, which adopts more or less the orientation of R289 in the proximal position, and interacts with flavin-bound sulfite. 7 The homologue of R289 is in the proximal position in most structures of family members.…”

“…Several residues in that proteasesensitive loop are invisible in all structures. 3,[5][6][7] There is evidence that residues of this loop interact with the active site during catalysis, since the mere proteolysis of one specific peptide bond in the loop induces alterations of the kinetic parameters. 9 Despite the amount of experimental data collected over the past decades, two mechanistic hypotheses (see Figure 3) remain in debate.…”