Crystal Structure of Cindoxin, the P450cin Redox Partner

Abstract

The crystal structure of the flavin mononucleotide (FMN)-containing redox partner to P450cin, cindoxin (Cdx), has been determined to 1.3 Å resolution. The overall structure is similar to that of the FMN domain of human cytochrome P450 reductase. A Brownian dynamics–molecular dynamics docking method was used to produce a model of Cdx with its redox partner, P450cin. This Cdx–P450cin model highlights the potential importance of Cdx Tyr96 in bridging the FMN and heme cofactors as well P450cin Arg102 and Arg346. Each of the single-site Ala mutants exhibits ∼10% of the wild-type activity, thus demonstrating the importance of these residues for binding and/or electron transfer. In the well-studied P450cam system, redox partner binding stabilizes the open low-spin conformation of P450cam and greatly decreases the stability of the oxy complex. In sharp contrast, Cdx does not shift P450cin to a low-spin state, although the stability of oxy-P450cin is decreased 10-fold in the presence of Cdx. This indicates that Cdx may have a modest effect on the open–closed equilibrium in P450cin compared to that in P450cam. It has been postulated that part of the effector role of Pdx on P450cam is to promote a significant structural change that makes available a proton relay network involving Asp251 required for O2 activation. The structure around the corresponding Asp in P450cin, Asp241, provides a possible structural reason for why P450cin is less dependent on its redox partner for functionally important structural changes.

Identification, characterization and molecular adaptation of class I redox systems for the production of hydroxylated diterpenoids

“…On a molecular level redox partner promiscuity of class I P450s is currently thought to be dependent on the amino acid decoration of the respective ferredoxin binding site. However, our current understanding of class I redox cascades is incomplete as only three class I P450s in complex with their respective innate ferredoxins have been structurally characterized [ 14 , 33 – 35 ].…”

“…In this instance, the combination of Pdx and Adr (mitochondrial adrenodoxin) allowed functional activation of P450cin, albeit in slow rates [ 36 ]. The recent crystal structure of the P450cin complex with its innate redox partner, a highly unusual FMN-containing flavodoxin (Cdx), demonstrated that P450cin does not depend on structural changes mediated by its redox partner protein [ 35 ]. Hence P450cin demonstrates a relaxed redox partner preference compared to P450cam.…”

Evolutionary Relationships Between Low Potential Ferredoxin and Flavodoxin Electron Carriers

“…For Flds and [2Fe-2S] Fds, organisms having a single PEC display a single mode with average lengths of ∼150 and ∼90 residues, respectively. These "short-chain" Flds and [2Fe-2S] Fds are well represented in the Protein Data Bank, including Desulfovibrio vulgaris Fld (2FX2) (Watt et al, 1991), Desulfovibrio desulfuricans Fld (3KAP) (Romero et al, 1996), Citrobacter braakii Fld (4OXX) (Madrona et al, 2014), Trichomonas vaginalis Fd (1L5P) (Crossnoe et al, 2002), Equisetum arvense Fd (1WRI) (Kurisu et al, 2005), and Scenedesmus fuscus Fd (1AWD) (Bes et al, 1999). In contrast, organisms having a single [4Fe-4S] Fd display two distinct modes.…”

Structural and catalytic properties of the peroxygenase P450 enzyme CYP152K6 from Bacillus methanolicus

“…Most P450s require electron transfer from one or more redox partner proteins in order to generate compound I. In bacterial systems these are typically soluble, NAD(P)H-dependent ferredoxin reductases and their cognate ferredoxins, although flavodoxins were also shown to support selected bacterial P450s [ [4] , [5] , [6] ]. In eukaryotes, the enzyme cytochrome P450 reductase ( CPR or POR), a natural fusion protein combining NADPH-ferredoxin reductase-like and flavodoxin-like domains, provides electrons for the function of the P450s [ 7 ].…”