Heme groups flavocytochrome

The success of Chapman and co-workers in expression of flavocytochrome 2 in E. coli [23] is encouraging in its impUcations for future expression of flavoproteins in this host because, in their experience both the flavin and heme groups are incorporated into the recombinant protein. Moreover, the bacterial expression system produces the protein 500-1000 fold more efficiently than the yeast from which it was cloned. The enzyme produced in E. coli, however, lacks the first five amino acid residues at its amino terminus, a result which presumably reflects subtle differences in protein synthesis between the two organisms. 

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... 

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... 

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 6. The Interaction of a Cytochrome c Molecule with a Single Flavocytochrome b Subunit as Predicted by Short et al. (1998). The two proteins are shown as ribbon diagrams. The heme groups are shown in stick representation. The interface region between the two proteins is indicated by the dotted line. 

Similarly, flavocytochrome C552 (Cyt C552, sulfide cytochrome c oxidoreductase), that includes FAD and two covalently linked heme groups, is bioelectrocatalytically active for the oxidation of sulfide to sulfur [13] ... 

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). 

One of a few cases in which electron transfer of redox enzymes is expressed directly and reversibly at an electrode is concerned with p-cresol methylhydroxylase (PCMH). This is a flavocytochrome c enzyme of 115 kDa, which catalyzes the oxidative hydroxylation of p-cresol to p-hydroxybenzyl alcohol and subsequently to p-hydroxyben-zaldehyde. The structure of PCMH has recently been determined (56) at 3 A resolution. It is an a2 2 tetramer, with one subunit containing a covalently bound FAD and the other containing a c-type heme group. 

Unlike the E. coli fumarate reductase, flavocytochrome Cj (Fee,) from the marine bacterium Shew one lla frigidimarina is a soluble enzyme. It has four heme groups instead of Fe-S clusters, and the FAD is not covalently bound, but otherwise the active site and tire mechanism are similar to the E. coll enzyme. Conventional methods for studying the active site are not useful here, as the electronic spectrum of the flavin is masked by the intense transitions from the four heme groups, and the one-electron radical (which should be EPR active) is inherently unstable. As discussed above, the instability of the radical means that two electrons transfer cooperatively and this makes it easy to observe the FAD by voltammetry. ... 

Whiehever mechanism operates, it is clear that the rate of reduetion of the flavin group is totally limited by the cleavage of the aC-H bond sinee the deuterium kinetic isotope effect for this step is around 8 (Miles et al., 1992 Pompon et al., 1980). However, in flavocytochrome 2 the rate of flavin reduetion is some 6-fold faster than the overall steady-state turnover rate (Daff et al., 1996a). As a consequence the flavin reduction step eontributes little to the rate limitation of the overall catalytic cycle (Figure 3). In faet it is eleetron transfer from flavin-semiquinone to b2 -heme that is the major rate-determining step and this is discussed in the following seetion. 

The crystal structures of Ffr from two Shewanella spp. and Ifc have been determined and are very similar to each other. The FAD domain of these flavocytochromes has significant structnral similarity to other FAD-binding proteins. The heme-binding domain shows very tittle secondary structure. All of the hemes are coordinated by two histidines and are in close distance to each other. Hemes 1 and 2 are positioned in a perpendicular motif, whereas hemes 2 and 3 are in a parallel stacked motif These three hemes can be superimposed to hemes 5 7 of HAO and 2 4 of NrfA (Figure 3). Heme 4 of Ffr is deviated from the corresponding heme 8 position in HAO, because it is oriented toward the FAD group. 

Fig. 3. The three-dimensional structure of Saccharomyces cerevisiae flavocytochrome 62 as determined by Xia and Mathews (25). (A) The C-terminal tails and flavin mononucleotide (FMN) and heme prosthetic groups are highlighted in this view, which is looking down the fourfold axis of symmetry. The four subunits are numbered 1 to 4 the shaded portions seen in the subunits labeled 2 and 4 represent the two heme domains, which are disordered in the structure. (B) A side view, perpendicular to view A, is also shown. 

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. 

Circular dichroism (CD) spectroscopy has been used to study the cleaved (73), deflavo (74), and intact (19, 60) forms of flavocytochrome 62 from S. cerevisiae and the intact enzyme from H. anomala (73, 75). Magnetic circular dichroism (MCD) has been used to probe the holo-and deflavo-cleaved enzyme from S. cerevisiae (76), as well as the cytochrome 62 core (77). It was noted that bands in the CD and MCD spectra ascribed to the heme were affected by removal of the FMN group, providing direct evidence of an interaction between the two prosthetic groups (73). Circular dichroism experiments on the intact and cleaved forms of S. cerevisiae flavocytochrome 62 led Jacq and Lederer (19) to conclude that there were differences in the heme environment between these two forms of the enzyme. 

EPR signals for both the flavosemiquinone radical and the low-spin ferric heme have been reported (65, 78-82). The flavosemiquinone signal, which is easily observed at 123 K, shows a typical g value of 2.0039 0.002 (65). The bandwidth, which is around 15 G, is very like that of an anionic, or red, semiquinone (65). The EPR signal of the low-spin ferric heme can be observed at low temperatures ( 28 K) and shows g values of 2.99, 2.22, and 1,47 (65), which are similar to those found for cytochrome 65 (81). EPR rapid freezing studies have allowed the amounts of semiquinone and ferric heme to be monitored during reduction of the enzyme by L-lactate (66). This has proved to be extremely useful in the development of kinetic schemes to describe the flow of electrons in the enzyme. The distance between the prosthetic groups in H. anomala flavocytochrome 62 has been estimated from EPR experiments and spin-lattice relaxation measurements (82). Pyruvate was used to stablize the flavosemiquinone and the effect on the signal of this species from oxidized and reduced heme was measured. The results indicated a minimum intercenter distance of 18-20 A (82). 

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