Heme-binding domain

Figure 8.1 Schematic of a typical cytochrome P450 protein showing the approximate locations for the heme binding domain, the proline-rich region and the I helix.

LS, K.H.D. Lederer, F. (1983). On the presence of a heme-binding domain homologous to cytochrome b5 in Neurospora crassa assimilatory nitrate reductase. The EMBO Journal 2, 1909-14. 

While the majority of biochemical studies have been conducted with the enzyme from Catharanthus over the last 3 decades, G10H was first cloned in Arabidopsis thaliana by employing a PCR strategy that used degenerate primers derived from the conserved heme-binding domain of P450s. From 16 clones obtained, one of the expressed proteins (CYP76C1) was shown to possess functional... 

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. 

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. 

The structure of this family of cyclases is distinct from the receptor type, the active enzyme being an a-/3 heterodimer of 76- and 80-kDa subunits, respectively (Kamisaki et al., 1986a Schmidt et al., 1993 Nakane and Murad, 1994). Although each subunit apparently contains catalytic and heme binding domains, enzyme activity is absolutely dependent on the presence of both subunits (Buechler et al., 1991 Schmidt et al., 1993 Nakane and Murad, 1994). The enzyme possesses a basal cyclase activity that is preferentially dependent on Mn, and activity is retained even in the absence... 

The mammalian Me cyts bs contain 134 amino-acid residues and show 93-98% sequence similarity. OM cyt bs (146 residues) is larger than Me cyts bs and shares only 68% sequence similarity with bovine Me cyt bs " Both Me cyts bs and OM cyt bs have three domains an N-terminal hydrophilic domain, a medial hydrophobic domain, and a C-terminal hydrophilic domain. In the cytosol, the 100-amino-acid N-terminal domain, which binds the A-type heme, participates in electron-transfer reactions.The heme-binding domains of Me cyt bs and OM cyt bs exhibit very similar protein folds. They have six a-helices and four /3-strands, two of which form a short /3-sheet. The heme is located in a pocket formed by four a-helices and a /3-sheet, with its propionates pointing towards the solvent and the opposite heme edge buried in a hydrophobic pocket. The planes of the heme axial His ligands are in a near-parallel orientation (Figure 10). 

Cytochrome contains 4 His residues in membrane-spanning helices that are conserved in the cyt b of the 6-Cj complex of mitochondria from many phyla as well as in photosynthetic bacteria, cyanobacteria, and chloroplasts (1-3). The hydropathy plots for the amino acid sequences of these cytochromes were highly correlated mathematically (1,4). This led to the prediction that the four His residues used to coordinate the two hemes of the cytochrome are located on two of the membrane-spanning helices, with a His pair coordinated to a heme on each side of the membrane thereby cross-linking the two helices. The cyt b protein of oxygenic photosynthesis which is --i the size of cyt b (6-Cj) constitutes the heme-binding domain of the latter. 

The sequence of the solrrble, negatively charged, heme-binding domain of microsorrtal cyts is highly conserved in eukaryotes with about 80% iderrtity and very corrservative srrbstitutions. The two most conserved motifs are the HPGG,... 

Figure 1. Comparison of heme binding domain and oxygen binding domain of the bell pepper HPO lyase and P450 consensus sequence, or the other P450s specialized for lipid peroxides.

Figure 1 The FixL/FixJ two-component regulatory system. In two-component regulatory systems, the inputs and outputs are variable, but the transmitter and receiver domains from unrelated systems share 20% identity. The input of the sensor FixL is a heme-binding domain that inhibits phosphorylation when O2 binds and that permits enzymatic activity when O2 leaves. The output response is transcriptional activation at nitrogen fixation promoters for only hyrpoxic conditions. (From Ref. 36.)...

Figure 5 Structures of the regulatory FG loop of the B. japonicum FixL heme-binding domain (5yFixLH) in the on and off states, as defined by measurements of autophosphorylation (enzymatic Scheme 1, Step 1). The figure shows an overlap of the refined models for both the unliganded on state (light gray), and the cyanide-boimd off state (dark gray), based on the crystal structures of met- and cyanomet-5yFixLH determined to 2.4-A and 2.7-A resolution, respectively. (From Ref 14.)...

A variety of structures have been solved for FixL heme-binding PAS domains (14,53,57). They include the initial crystal structure of the B. japonicum FixL heme-PAS domain in both on (unliganded) and off (cyanide-bound) conformations, as defined by measurements of autophosphorylation (enzymatic Scheme 1, Step 1) (14). The structures of the on Fe and Fe forms of the S. meliloti FixL heme-binding domain and additional off Fe 02 and Fe imidazole forms of the B. japonicum FixL heme-binding domain are also available (53,57). Based on the structures of the ferric unliganded (Fe , or met) and cyanide-bound (Fe CN , or cyanomet) forms of the heme-PAS domains, a small ligand-induced conformational change was postulated to cause the kinase inhibition (14) (Fig. 5). 

We have obviated the need for isolating nucleating agents fix>m the parasite by utilizing an entirely synthetic dendrimeric peptide bionucleating template (BNTII) based on the putative heme binding domain of HRP II that functions as... 

The heme-binding domain of several proteins [cytochrome 65, flavocyto-chrome 2 > and sulfite oxidase designated as the cytochrome 65 fold (Guiard and Lederer, 1979)] have also great sequence homologies. These three proteins are probably the products of divergent evolution from a common ancestor. 

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