Sulfite Binding to a Flavodehydrogenase, Cytochrome b₂ from Baker's Yeast

Abstract

Baker's yeast L-lactate dehydrogenase (flavocytochrome b2) is a typical flavodehydrogenase, in that it accepts two electrons from the substrate but has a monoelectronic acceptor. Yet it forms a red semiquinone [Capeillere Blandin et al. Eur. J. Bioclwm. 54, 549-566 (1975)j and it is shown in this paper that it forms a reversible covalent complex with sulfite (Kd = 1.4 pM). This complex can be observed by difference spectroscopy and provides a convenient tool for visualizing the flavin chromophore, usually hidden behind the intense heme absorbance. A number of anions (D-lactate, oxalate and pyruvate) are inhibitors of the enzymatic reaction and induce spectral perturbations of the flavin spectrum. It is concluded that probably two positive charges exist at the active site: one which stabilizes the red semiquinone and one which attracts organic anions and sulfite. It is also concluded that the correlation between reactivity with sulfite and reactivity with oxygen among flavoproteins may not be as general as previously proposed [Massey et al. J. Biol. Chem. 244,[3999][4000][4001][4002][4003][4004][4005][4006] (1 969)].Free reduced flavin is rapidly reoxidized by oxygen, whereas enzyme-bound flavin either keeps or loses this property, according to the function of the protein. This phenomenon provides the basis for the now classical distinction, among flavoproteins, between oxidases and dehydrogenases. It is clear that the protein moiety plays a fundamental role in modulating the reactivity of the bound flavin, but this role is at present poorly understood, in spite of numerous efforts.A few years ago, Massey et al. [l] proposed an interesting correlation between the reactivity of flavoproteins toward oxygen and toward sulfite. It was shown that a number of flavoproteins were bleached by sulfite, which reversibly formed a covalent bond with N-5 of the flavin nucleus [2]; those proteins that reacted were oxidases and formed anionic (red) semiquinones; those that did not react were dehydrogenases and formed neutral (blue) semiquinones. A tentative explanation was presented, based on these and other observations: a positive charge at the active site of oxidases would facilitate sulfite binding and stabilize the anionic semiquinone ;

The Role of a β Barrel Loop 4 Extension in Modulating the Physical and Functional Properties of Long‐Chain 2‐Hydroxy‐Acid Oxidase Isozymes

“…In contrast, in the near-ultraviolet and visible regions, significant amplitude differences were observed, in particular between the maximum at 290 nm and the minimum at 270 nm. The sharp negative signal at 270 nm was observed before in other flavoproteins, in particular in tlavocytochrome b2, where its intensity was unaffected by the enzyme redox state, but was sensitive to the covalent addition of sulfite at the FMN N5 position (Lederer, 1978). There has been some debate as to the origin of this signal (Lederer, 1978;Tsong and Sturtevant, 1969;Sturtevant and Tsong, 1969;Iwatsubo and Risler, 1969).…”

“…This is, in particular, the case for glycolate oxidase (Macheroux et al, 1991), lactate oxidase (Massey et al, 1969) and flavocytochrome b, (Lederer, 1978). Sulfite binding to the prosthetic group can be monitored either spectrally, since it results in a reduced-type flavin spectrum, or kinetically, since it behaves as a competitive inhibitor.…”

Flavin–protein interactions in flavocytochrome b2 as studied by NMR after reconstitution of the enzyme with 13C‐ and 15N‐labelled flavin

“…The FMN content of the protein was determined either by complete denaturation of the protein in 6 m guanidine HCl and subsequent fluorescence measurement [6] or by difference spectrophotometry between the free oxidized and the sulfite‐complexed states [7]. The extinction coefficients for the difference measurements was ε 454 = 10.5 m m −1 ·cm −1 [7]. The concentration of free FMN was determined by spectrophotometric measurements, using ε 445 = 12.5 m m −1 ·cm −1 as absorption coefficient.…”

Inhibition of L‐lactate: cytochrome‐c reductase (flavocytochrome b2) by product binding to the semiquinone transient

“…Previously it has been shown for the S. cerevisiae flavocytochrome b2 [16] that pyruvate and other inhibitors are able to induce a perturbation of the oxidized enzyme spectrum at the level of the flavin absorbance (520 -420 nm). We report in Fig.…”