Aluminum‐substituted heme domain of P450BM‐3 (BMP): Introducing a heme‐derived fluorescent probe for studies of substrate binding and protein–protein interactions in cytochromes P450

Abstract

We describe a novel technique for heme removal and replacement in the heme domain of P450BM-3 (BMP). The method was applied to obtain the aluminum-protoporphyrin IX (Al-PP) substituted derivative of BMP (Al-BMP). The overall yield of the purified Al-BMP was about 15% as related to the initial amount of the hemeprotein. Al-BMP possesses extensive fluorescence in the 550-650 nm region with excitation in the porphyrin absorbance bands. The protein was shown to bind substrates of P450BM-3 (palmitic, arachidonic, and cis-parinaric acids) with affinities similar to those of the native enzyme (3-6 μM). However, the substrate-induced changes in fluorescence of Al-PP reveal the existence of a second, low-affinity substrate-binding site, which cannot be detected by the spin shift in the native, heme-containing BMP. Using fluorescence resonance energy transfer, we have demonstrated that Al-BMP forms a complex with the flavoprotein domain of P450BM-3 labeled with 7-ethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin maleimide, revealing the affinity similar to that of native BMP (K d = 5 μM at 0.06 M ionic strength). Therefore, aluminum-substituted BMP may serve as a valuable tool in studies on the mechanisms of interactions of P450s with their substrates and protein partners.

Kristalle in Minutenschnelle: Sofortige Mikrokristallisation verschiedenster Varianten der CYP102A1‐(P450BM3)‐Hämdomäne

“…Obwohl es durchaus Berichte gibt, welche die Rekonstitution von P450BM3 mit künstlichen Kofaktoren beschreiben (Aluminiumprotoporphyrin IX, Eisen (III)‐deuteroporphyrin IX und Methyliridiumdeuteroporphyrin IX), besteht ein Mangel an P450BM3‐Kristallstrukturen mit künstlichen Kofaktoren. Bis vor Kurzem wurde lediglich eine Mutante von P450BM3 mit Eisen(III)‐deuteroporphyrin IX in der PDB gemeldet (PDB ID: 5JQU und 5JQV) .…”

Crystals in Minutes: Instant On‐Site Microcrystallisation of Various Flavours of the CYP102A1 (P450BM3) Haem Domain

“…Although reports describing the reconstitution of P450BM3 with artificial cofactors (aluminium protoporphyrin IX, iron(III) deuteroporphyrin IX, and methyliridium deuteroporphyrin IX) do exist, there is a lack of P450BM3 crystal structures containing artificial cofactors, and, until recently, only the crystal structure of mutant P450BM3 with iron(III) deuteroporphyrin IX had been reported on the PDB (PDB ID: 5JQU and 5JQV) . Recently, our group reported the crystal structure of the P450BM3 haem domain incorporating manganese protoporphyrin IX (MnPPIX), at a mediocre resolution of 3.10 Å (PDB ID: 5ZIS), which, although sufficient to indicate the correct reconstitution of P450BM3 with MnPPIX, was insufficient to allow the discussion of structural perturbations of the side chains.…”

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

“…Requires exogenous fluorophore with high quantum yield CYP, GST Greinert et al, 1979Gut et al, 1985;Schwarz et al, 1993;Gorovits and Horowitz, 1995;Lim et al, 1995;Lakowicz, 1999 , 1999, 2001Kuznetsov et al, 2004;Shimada et al, 2005;Pearson et al, 2006;Archakov and Ivanov, 2011;Martin et al, 2015;Yablokov et al, 2017 Quartz crystal microbalance conductometric Davydov et al, 2013b;Spera et al, 2013 X-ray crystallography with traditional site-directed mutagenesis techniques to obtain answers to structural questions that might not be readily available by either technique alone. X-ray crystallography has also been useful for dissecting the protein-protein interactions of another CYP electron transfer partner, b 5 .…”

“…Therefore, the aluminum substitution was unlikely to have a major effect on the overall structure of the active site. Additionally, while the aluminum atom is not able to donate or accept electrons, it imparts a high degree of fluorescence to the protoprophyrin, thereby introducing an internal probe which allowed the authors to monitor the interactions between CYP P450 BM-3 and its substrates and protein partners using fluorescence energy transfer (Davydov et al, 2013b). The CYP P450 BM-3 retained its ability to interact with the reductase domain despite the substitution in the protoprophyrin and the newly imparted fluorescence from the aluminumprotoporphyrin IX allowed the authors to determine affinity between the CYP domain and the reductase domain.…”