Flavocytochrome electron acceptors

Flavin mononucleotide, 3absorption coefficients, 36 270 active site, 36 265-267 catalysis and electron transfer, 36 275-287 carbanion mechanism, 36 277-282 electron acceptors, 36 285-287 electron transfer pathway, 36 275-276, 282-285... 

PCMH is a flavocytochrome c localized in the periplasmic space of several types of Pseudomonad (Hopper and Taylor, 1977). It catalyzes the oxidation of / -cresol first to / -hydroxybenzyl alcohol and then to / -hydroxybenzaldehyde. Electrons are passed sequentially to the endogenous cytochrome subunit and then to an exogenous secondary electron acceptor protein, possibly an azurin or another cytochrome (Causer et al., 1984). 

The physiological pathway of electron transfer in flavocytochrome is from bound lactate to FMN, then FMN to 52-heme, and finally 52-heme to cytochrome c (Fig. 9) (2,11, 80,102). The first step, oxidation of L-lactate to pyruvate with concomitant electron transfer to FMN, is the slowest step in the enzyme turnover (103). With the enzyme from S. cerevisiae, a steady-state kinetic isotope effect (with ferricyanide as electron acceptor) of around 5 was obtained for the oxidation of dl-lactate deuterated at the C position, consistent with the major ratedetermining step being cleavage of the C -H bond (103). Flavocytochrome 52 reduction by [2- H]lactate measured by stopped-flow spectrophotometry resulted in isotope effects of 8 and 6 for flavin and heme reduction, respectively, indicating that C -H bond cleavage is not totally rate limiting (104). 

Cytochrome c has often been used as an external electron acceptor in steady-state experiments with flavocytochrome 62 and usually gives rise to lower specific activities than would be found if ferricyanide was used as acceptor. The differences between cytochrome c and ferricyanide as electron acceptors are discussed further in the next section. Values of Km for cytochrome c from steady-state measurements are very dependent on the nature of the buffer used, with values ranging from 10 fj-M (10 mM Tris-HCl, pH 7.5,1 = 0.10 M NaCl) 134) to 180 fj.M (100 mM phosphate, pH 7.2) 135) seen with intact flavocytochrome 62 from S. cerevisiae. It is perhaps not surprising that the Km for cytochrome c is markedly affected in phosphate buffer considering the propensity for phosphate to bind to cytochrome c 136). 

Ferricyanide is the most commonly used electron acceptor in steady-state kinetic experiments on flavocytochrome 62. How is ferricyanide reduced by the enzyme Ogura and Nakamura suggested that ferricyanide could accept electrons only from the 62 heme (79). This is clearly incorrect, because dehemoflavocytochrome 62 and the isolated flavode-hydrogenase domain can still function as ferricyanide reductases, though at somewhat lower efficiency 51, 126). These results imply that ferricyanide can accept electrons from both flavohydroquinone and flavosemiquinone as well as heme. In heme-free cleaved enzyme from S. cerevisiae it was calculated that ferricyanide was reduced around 20 times faster by flavosemiquinone than by flavohydroquinone 126). This would mean that in the holoenzyme, reduction of ferricyanide would occur rapidly from heme and flavosemiquinone. The fact that ferricyanide is reduced by both 62 heme and flavosemiquinone, and that cytochrome c is reduced only by 62 heme, might be an explanation for the observation that specific activities of the enzyme determined with cytochrome c are usually somewhat lower than those determined with ferricyanide. 

Flavocytochrome hi can be cleaved by controlled proteolysis. Each protomer is folded into two domains having different functions, the L-lactate dehydrogenase, the flavodehydrogenase (FDH)4, a tetramer of molecular mass of 160 kDa, and its electron acceptor, the cytochrome hi core, a monomer of molecular mass of 13 kDa (Gervais et al. 1980), which then acts as a one-electron donor to cytochrome c. ... 

Electronic absorption spectroscopy charge transfer transitions, 19 71 d-d transitions, 19 70, 71 flavocytochrome b, 36 269-271 intraligand transitions, 19 71-80 of organometallics, 19 69-80 Electronic coupling, between donor and acceptor wave functions, 41 278 Electronic nuclear double resonance spectroscopy, molybdenum center probes, 40 13... 

In contrast to the flavin oxidases, flavin dehydrogenases pass electrons to carriers within electron transport chains and the flavin does not react with 02. Examples include a bacterial trimethylamine dehydrogenase (Fig. 15-9) which contains an iron-sulfur duster that serves as the immediate electron acceptor167 169 and yeast flavocytochrome b2, a lactate dehydrogenase that passes electrons to a built-in heme group which can then pass the electrons to an external acceptor, another heme in cytochrome c.170-173 Like glycolate oxidase, these enzymes bind their flavin coenzyme at the ends of 8-stranded a(i barrels similar... 

All these studies indicate that electron transfer within the flavocytochrome -cytochrome c complex is dependent upon a number of factors such as the distance between donor (cytochrome b2 core or TNS) and acceptor (cytochrome c). their relative orientation, their chemical nature and the structure of the protein medium involved in the electron transfer. 

Yeast mitochondrial flavocytochrome 2 (lactate cytochrome c oxido-reductase) catalyzes the transfer of electrons from L-lactate to various acceptors, cytochrome c being the physiological acceptor. The protoheme and flavin mononucleotide, because of their higher redox potential, can both be reduced completely by L-lactate the enzyme accepts a total of three electrons per protomer, which amounts to twelve electrons for the stable active tetramer. Both types of prosthetic group are quantitatively reoxidized by external acceptors. 

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