Flavocytochrome cytochrome domain

FIGURE 7. Stereo diagram of one subunit of flavocytochrome b. Only residues ln486 are shown, the remainder being involved in intermolecular interactions. The flavin-binding domain is at the top and the cytochrome domain is at the bottom. The flavin and heme groups are shown as skeletal models. 

FIGURE 2. A Subunit of S. cerevisiae Flavocytochrome bj The protein is shown as a ribbon diagram with the heme and flavin cofactors in a stick representation. The two domains are clearly delineated. Cyt, cytochrome domain Flav, flavin domain H, interdomain hinge peptide C, C-terminal tail. 

The characterisation of the complexation between flavocytochrome b2 and cytochrome c has been the subject of many studies (see for example Short et al., 1998 Daff et al., 1996b and CapeillEre-Blandin, 1995). Work on the anomala flavocytochrome b2, for which there is no crystal structure, led to the conclusions that the cytochrome c binding site involved both the flavodehydrogenase and cytochrome domains (CapeillEre-Blandin and Albani, 1987) and that the complex was stabilised by electrostatic interactions (CapeillEre-Blandin, 1982). It is clear that similar conclusions hold true for the S. cerevisiae enzyme (Daff et al., 1996b) for which the crystal... 

Labeyrie, F., Beloeil, J. C., and Thomas, M. A., 1988, Evidence by NMR for mobility of the cytochrome domain of flavocytochrome bi, Biochim. Biophys. Acta. 953 134nl41. 

Labeyrie et al. (41) isolated a trypsin fragment of 11 kDa from S. cerevisiae flavocytochrome 62 that contained heme but was devoid of flavin and had no lactate dehydrogenase activity. The fragment, which was referred to as cytochrome 62 core, was found to have spectral properties very like those of microsomal cytochrome 65 (41). This similarity with cytochrome 65 is borne out by comparisons of amino acid sequence (42-44). The sequence similarity extends to related heme domains of sulfite oxidase (45, 46) and assimilatory nitrate reductase (47). The existence of a protein family with a common cytochrome 65 fold was suggested by Guiard and Lederer (48) and this is supported by the close similarity between the three-dimensional structures of microsomal cytochrome 65 (49) and the cytochrome domain of flavocytochrome 62 (23-25). 

The cytochrome 62 core has been shown by amino acid sequence determination to be located at the N-terminus of the flavocytochrome 62 polypeptide chain (43). It is clearly seen as a distinct domain in the crystal structure, in close contact with the much larger FMN-contain-ing domain (23-25) (Fig. 5). The cytochrome domain consists of resi-... 

The reduction potentials for the heme and FMN prosthetic groups of flavocytochromes 62 from S. cerevisiae and H. anomala are listed in Table I. Values for various modified forms of the enzyme, such as the flavin-free (deflavo) derivative, and the isolated cytochrome domain (the cytochrome 62 core) are also reported in Table I (64-69). The reduction potentials for the heme group are as expected for a 65-type cytochrome (70), with little difference in the values for different forms of protein, e.g., the deflavo-derivative of the holoenzyme and the isolated cytochrome 62. The reduction potentials for the FMN group are not too different from those of the heme (about 50 mV difference), consistent with reversible electron transfer between the two prosthetic groups (10). 

Abbreviations Sx, the cleaved form of flavocytochrome 62 from S. cerevisiae deflavo-Sx, S , in which the FMN prosthetic group has been removed Sx-62-core, the isolated cytochrome domain (or core) of Sx Hj, the intact flavocytochrome 62 from H. anomala deflavo-Hi, Hi in which FMN has been removed and Hi-62-core, the isolated heme domain (or core) of Hi. 

NMR at 400 MHz has been used to probe the mobility of the cytochrome domain within the holoenzyme (84). The linewidths of heme resonances downfield of 4-12 ppm and upheld of -4 ppm have been compared for the holoenzyme and the cytochrome 62 core and indicate that the cytochrome domains of the holoenzyme are markedly mobile (84). NMR has also been used to investigate the nature of the interaction between flavocytochrome 62 and cytochrome c (85,86) with the ultimate aim of defining the binding site on flavocytochrome 62 used by its physiological partner. The extensive NMR studies on the interaction between cytochrome 65 and cytochrome c (87,88) provide a useful background to the work with cytochrome 62 ... 

It is quite clear that protein engineering will contribute substantially to future investigations of electron transfer in flavocytochrome 62. To date protein engineering has been used to generate a number of single amino acid substitutions and has allowed the independent expression of the two functionally distinct domains of the enzyme. These two approaches can be readily combined, for example, to express the flavodehydrogenase domain with an active site mutation, thereby sim-plyfing analysis of electron transfer to FMN without interference from the cytochrome domain. 

Some metalloflavoproteins contain heme groups. The previously mentioned flavocytochrome b2 of yeast is a 230-kDa tetramer, one domain of which carries riboflavin phosphate and another heme. A flavocytochrome from the photosynthetic sulfur bacterium Chromatium (cytochrome c-552)279 is a complex of a 21-kDa cytochrome c and a 46-kDa flavoprotein containing 8a-(S-cysteinyl)-FAD. The 670-kDa sulfite reductase of E. coli has an a8P4 subunit structure. The eight a chains bind four molecules of FAD and four of riboflavin phosphate, while the P chains bind three or four molecules of siroheme (Fig. 16-6) and also contain Fe4S4 clusters.280 281 Many nitrate and some nitrite reductases are flavoproteins which also contain Mo or... 

Flavocytochrome b2 from Saccharomyces cerevisiae, a member of the FMN-dependent oxidoreductase superfamily, catalyzes the two-electron oxidation of lactate to pyruvate with subsequent electron-transfer to cytochrome c via the bound flavin [55], What distinguishes the enzyme from other family members is the N-terminal fusion of a heme-binding domain to the ySa-barrel structure, which hosts the primary active site. Rather than dumping the electrons from the reduced flavin hydroquinone onto molecular oxygen, they are transferred intramolecularly to the heme-binding domain and from there in a second intermolecular step to cytochrome c. 

Flavocytochrome c3 (EC 1.3.99.1) isolated from Shewanella frigidimarina is a unique fumarate reductase of 63.8 kDa MW in a single subunit composed of two domains. The active site is located in the flavin domain. The heme domain contains four c-type hemes, each with a bis-His axial ligation. It has been proposed that this domain is similar to cytochrome c3 from Desulfovibrio desulfuricans. On pyrolytic graphite (edge) electrodes in the presence of polymyxin the single redox centres were examined. Fumarate addition is followed by a catalytic current [108]. 

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. ... 

This leads us to the following conclusion the flavodehydrogenase domain in the native flavocytochrome hi exerts very little influence on the efficiency of the electron transfer between cytochrome b2 and cytochrome c within the complex. This result implies that similar transition states must be achieved in the reactions of cytochrome c with both free and bound cytochrome b2 Consequently, the electron transfer occurs via the same mechanism involving the same pathway through the intra-molecular reaction complex. Thus, in both flavocytochrome b2 - cyt.c and cyt.b2 core - cyt.c complexes, the relative distance and orientation of the heme b2 and heme c planes are optimal to achieve the electron-transfer process. 

Addition of cytochrome hi core to a solution of TNS does not modify the fluorescence of the probe. However, upon addition of the cytochrome to a solution of FDH-TNS complex, an increase in TNS fluorescence was observed, as the result of an increase in the affinity between the probe and the flavoprotein (A.ex, 320 nm) (Albani, 1993). X-ray diffraction studies have indicated that FMN is buried in the flavin-binding domain of flavocytochrome hi (Zia et al. 1990), and fluorescence studies have shown that binding of TNS to the FDH does not induce any release of the flavin from its binding site (Albani, 1993). Titration of a constant concentration of FDH-TNS complex with cytochrome hi core yields a sigmoidal curve for the TNS intensity increase (Fig. 4.18) (Albani, 1997). Thus, interaction between cytochrome hi core and FDH is cooperative. 

Noble MA, Girvan HM, Smith SJ, Smith WE, Mu-rataliev M, Guzov VM, Feyereisen R, Munro AW (2007) Analysis of the interactions of cytochrome bj with flavocytochrome P450 BM3 and its domains. Drug Metab Rev 39 599-617... 

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