Heme proteins flavocytochrome

The folding pattern of cytochrome b5 is also found in the complex heme protein flavocytochrome b2 from yeast (Chapter 15)133 and probably also in liver sulfite oxidase134,135 Both are 58-kDa peptides which can be cleaved by trypsin to 11-kDa fragments that have spectroscopic similarities and sequence homology with cytochrome b5. Sulfite oxidase also has a molybdenum center (Section H). The 100-residue N-terminal portion of flavocytochrome b2 has the cytochrome b5 folding pattern but the next 386 residues form an eight-stranded (a / P)8 barrel that binds a molecule of FMN.133,136 All of these proteins pass electrons to cytochrome c. In contrast, the folding of cytochrome... 

Since the primary structure of a peptide determines the global fold of any protein, the amino acid sequence of a heme protein not only provides the ligands, but also establishes the heme environmental factors such as solvent and ion accessibility and local dielectric. The prevalent secondary structure element found in heme protein architectures is the a-helix however, it should be noted that p-sheet heme proteins are also known, such as the nitrophorin from Rhodnius prolixus (71) and flavocytochrome cellobiose dehydrogenase from Phanerochaete chrys-osporium (72). However, for the purpose of this review, we focus on the structures of cytochromes 6562 (73) and c (74) shown in Fig. 2, which are four-a-helix bundle protein architectures and lend themselves as resource structures for the development of de novo designs. 

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. 

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. 

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. 

Flavocytochrome Fumarate Reductase. The flavocytochrome fumarate reductase (Fff) is a soluble periplasmic protein from Shewanella spp. that reduces fumarate but does not oxidize succinate, in contrast to the membrane-bound fumarate reductases that are related to succinate dehydrogenases, and transfer electrons from quinol to fumarate. It is a monomeric protein of 63.8 kDa that is composed of three domains. The N-terminal domain contains four c-type hemes, and the flavin domain contains noncovalently bound FAD and is related to flavoprotein subunits of membrane-bound fumarate reductases and succinate dehydrogenases. There is also a third domain in the flavocytochromes that has considerable flexibility and may be involved in controlling access of substrate to the active site. The macroscopic redox potentials of the fom hemes of Ffr are —102, —146, —196, and -23 8 mV, while that of FAD is —152 mV. The low redox potential of FAD in Ffr compared to that in membrane-bound fumarate reductase (—55 mV) may explain why it is unable to oxidize succinate. 

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. 

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 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). 

The E. coli harboring a plasmid, pDS-b2 (Fig. 16), designed for in vitro transcription and translation, were noticeably pink in color 147). This results from constitutive expression of flavocytochrome 62 at up to 5% of the total soluble protein in these cells. Expressed enzyme contains full stoichiometric amounts of FMN and heme 147). Expression of flavocytochrome 62 in vivo was not expected with this vector because there is no E. coli ribosome-binding site (rbs). It appears that a fortuitous rbs exists within the region of DNA encoding the mitochondrial targeting sequence and this leads to initiation of translation at Met 6 of the mature flavocytochrome 62 147). The absence of residues 1-5... 

Another Class II P-450 redox system that has been extensively studied is the cytosolic flavocytochrome P-450 BM3 from Bacillus megaterium. BM3 is the fusion of a soluble P-450 domain with CPR." " " The FAD of BM3 is reduced by NADPH the electrons are transferred to FMN and then finally to the substrate-bound P-450 domain." " BM3 is the fastest reported P-450 monooxygenase,with the rate of hydride transfer from NADPH to FAD and the rate of electron transfer from FMN to heme several-fold above the mammalian P-450 redox systems." The structure of the full-length protein has yet to be solved, but the structure of the FAD- and NADPH-binding domain has been determined." This domain closely resembles rat liver CPR and contains several conserved residues implicated in NADPH binding and flavin reduction. 

In another study, we investigate electron transfer between flavocytochrome b2 and cytochrome c in presence of TNS. In principle, in absence of FMN or heme, electron transfer cannot occur between flavocytochrome b2 and cytochrome c. 2-p-toluidinylnaphthalene-6-sulfonate (TNS) is a fluorescent probe used to study the interaction between proteins, proteins and their ligands and to follow the global and local motions in proteins. TNS binds to the flavocytochrome b2 and to the flavodehydrogenase. 

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