FMN domain

Fig. 12. Crystal structure of the complex formed between the heme and FMN domains of cytochrome P450BM-3 133). The FMN domain docks on the proximal surface of the heme domain. The thicker trace in the heme domain highlights residues 387 to the heme ligand, Cys400. This section of polypeptide contacts the FMN and might provide an electron transfer conduit to the heme ligand. The two interacting surfaces are electrostatically complementary, with similar complementarity expected for HO and NOS.

Utilization of a domain linker to control electron flow is not unique to NOS. Like NOS, P450BM-3 has the heme and reductase domains fused to give a heme-FMN-FAD architecture (75). In addition, the linker between the heme and FMN domains is critical for electron transfer. Engineering studies on the P450BM-3 linker reveals that the length of the linker but not the sequence is critical in controlling the FMN-to-heme electron transfer reaction 135,136). Similar experiments with flavocy-tochrome b2 137) illustrate the importance of the linker in interdomain electron transfer, presumably by assisting in proper orientation of redox partners. The same appears to be true for NOS, with the important... 

Fig. 13. Schematic representation of the overall NOS architecture and summary of work presented in 139). Heterodimers were generated to test if electron transfer from the FMN domain proceeds via an inter- or intrasubunit process. When holo-NOS containing an inactive heme domain was dimerized with an active heme domain, activity was observed. However, when active holo-NOS was dimerized with the inactive heme domain, no activity was observed. These results indicate that the flavin domain of monomer A transfers electrons to the heme domain of monomer B.

The interaction of the BM3/FMN domain with the BMP domain involves four segments of the former and three segments of the latter (Figure 5). The largest such segment of BM3/FMN is the pi to al loop and... 

The structure of CPR is significant because this membrane-bound protein mediates electron transfer from NADPH to all microsomal P450 enzymes. A soluble form of CPR was generated by limited protease digestion and crystallized. The structure consists of a N-terminal FMN domain linked to... 

Figure 7 The crystal structure of NADPH-cytochrome P450 reductase (CPR). The FAD and FMN domains are linked by a hinge domain...

Figure 3.11. Structure of complex formed between the heme and FMN domains in P450BM3. The FMN domain docks into the concave depression on the heme domain proximal surface.

Figure 3.12. The crystal structure of P450 reductase. Note that the FMN and FAD are in direct contact. The schematic diagram illustrates that it may be necessary for the FMN and FAD domains to separate to enable the FMN domain to dock onto the P450 prior to the FMN-to-heme electron transfer reaction.

Figure 3.13. A close-up view of the interface formed between the heme and FMN domains in the P450BM3 electron transfer complex. The heme domain is more darkly shaded. There are only two direct H-bonds between the two domains Hisl00(heme)-Glu494(FMN) and Asnl01(heme)-At 498(FMN). The remainder of the electrostatic interactions are formed by bridging water molecules. Note that the FMN directly contacts the heme domain near Gln387. The section of polypeptide leading from Gln387 to the Cys400 ligand and heme could provide a selective electron transfer conduit.

The FMN domain, consisting of residues from 67 to 231 of rat POR, is structurally very similar to the bacterial flavodoxins and consists of a five-stranded parallel p-sheet flanked by five a-helices (Fig. 2.2), with the FMN located at the tip of the C-terminal side of the P-sheet. In addition to the binding site for the FMN prosthetic group, this domain contains residues mediating binding of and electron transfer to acceptors such as cyt c and P450. FMN is relatively loosely boimd 10 M) and can be reversibly removed from the enzyme by high salt treatment [27,49]. In the absence of FMN, electron transfer to all acceptors, with the exception of fenicyanide, is abolished. 

Fig. 2.5 Top panel, a Model of a complex between P450 and NADPH-cytochrome P450 oxidoreductase (FOR). A complex of P450 (red) and Mol A of the hinge-deletion mutant of POR(ATGEE), denoted as PORT e [53]). the flavin mononucleotide (FMN) domain (blue) and flavin adenine dinucleotide (FAD) domain (yellow)] and an enlarged view showing the relative orientation of the EMN and heme, b and c Open-book representation of molecular surface at the interface of P450 (b) and the EMN domain of POR (c). Five salt-bridge pairs are shown with same let-...

S Role of the FMN Domain and Connecting Domain in the Cytochrome P450 Interaction... 

The FMN domain has conserved patches of acidic residues involved in the electrostatic interactions with its electron transfer partners, and these interactions are specific for each electron transfer partner. Cross-linking experiments... 

---