SchuenemannCytochromeP450-ExtraMaterialsschuenemanncytochromep450-额外的材料

Titelmasterformat durch Klicken bearbeiten,Textmasterformate durch Klicken bearbeiten,Zweite Ebene,Dritte Ebene,Vierte Ebene,Fnfte Ebene,*,V.Schnemann,Technische Universitt Kaiserslautern,Germany,C.Jung,KKS Ultraschall AG,Switzerland,A.X.Trautwein,Universitt zu Lbeck,Germany,INTERMEDIATES IN THE REACTION CYCLE OF CYTOCHROME P450cam,Motivation,The physiologically important enzyme superfamily cytochrome P450 catalyzes a variety of reactions,such as aliphatic and aromatic hydroxylations,epoxidations,heteroatom oxidation,and N-and O-dealkylation,by transfer of an active oxygen atom from its heme unit to the substrates.All enzymes of the cytochrome P450 family have a hydrophobic binding pocket,in which resides a protoporphyrine IX with its iron center being coordinated to an axial cysteine ligand 1.,The enzyme cytochrome P450cam from,Pseudomonas putida,hydroxylases(1R)-camphor as natural substrate and is regarded to be a representative enzyme for the whole P450 family.Mssbauer spectroscopic studies on this enzyme have been performed by Debrunner and coworkers in the seventies 2 of the last century.The results of these studies provided insight into the catalytic mechanism of the enzyme even before the crystal structure of the resting state of P450cam was published 3.,H,O,Fig.1 Cytochrome P450cam(left)adds an oxygen atom O at the 5-exo position of camphor(C,10,H,16,O,right).,Fig.2 The reaction cycle of cytochrome P450.The,“shunt“reaction used in this study is depicted in red.,The cytochrome P450 reaction cycle,The postulated enzymatic reaction cycle is shown in Fig.2.In the resting state of the enzyme the catalytically active heme iron center acquires the ferric low-spin state(S=1/2).After binding of the substrate camphor to the amino acid residue Tyr96 inside the heme pocket,the iron changes from the ferric low-spin to the ferric high-spin state(S=5/2).The transfer of the first electron originating from NAD(P)H via redox proteins(flavin and iron-sulfur proteins)reduces the iron to the ferrous high-spin state(S=2).,Subsequent binding of molecular oxygen to the iron forms a diamagnetic FeO,2,center,similar to the oxygenated state of myoglobin.The transfer of a second electron initiates catalytic steps which lead to an,iron-oxo intermediate called compound I(cpd I).,It is this intermediate which inserts the active oxygen atom into the substrate camphor.,The nature of the intermediate cpd I,Neither the electronic structure of cpd I of P450cam nor of any other P450 has been unambiguously determined up to now,but it is generally assumed that the putative cpd I in cytochrome P450 should contain the same electron and spin distribution as is observed for cpd I of peroxidases 4,catalases and many synthetic cpd I analogues 5.In these systems one oxidation equivalent resides on the Fe(IV)=O unit(d,4,S=1)and one is located on the porphyrin(S=1/2),constituting a magnetically coupled ferryl iron-oxo porphyrin,-cation radical system.For other enzymes like cytochrome c peroxidase a ferryl Fe(IV)coupled to an organic radical on the protein(S,Radical,=1/2)has been proposed 6.,R-H+,O,2,+2e,-,+2H,+,R-,O,H+H,2,O,S,Fe(IV),=1 S,Radical,=1/2,Fig.3 The Intermediate cpd I(left)inserts oxygen into the substrate R-H via the reaction shown on the right.,Experimental techniques,Reaction intermediates can be trapped by rapid freeze-quench experiments(Fig.4).This technique allows to prepare samples for Mssbauer-and electron paramagnetic resonance(EPR-)spectroscopy.In order to detect cpd I in cytochrome P450cam we have used the“shunt reaction(see Fig.2)with peroxy acetic acid.,Freezing,of the,reaction,mixture,within,milliseconds,Addition of,peroxy,acetic,acid,to native,Cyt,P450,cam,Investigation with,Mssbauer,Spectroscopy,Multi-Frequency,EPR,Spectroscopy,Fig.4,The start:Mssbauer Spectroscopy on camphor-free cytochrome P450cam:,The Mssbauer spectrum obtained at 4.2K and B=20 mT of cytochrome P450cam without the substrate camphor is shown in Fig.5.The observed magnetic splitting and the corresponding EPR spectrum(Fig.6)are consistent with a ferric low-spin state of the iron.The Mssbauer spectrum has been analyzed by the spin-Hamiltonian formalism(solid line in Fig.5)and the parameters are shown in Tab.1 7.,S,(mm/s),E,Q,(mm/s),g,A,/g,N,N,(T),1/2,0.38,2.85,-1.8,(1.91,2.26,2.45),(-45,10,19),Fig.5,Fig.6,Table 1.Mssbauer parameters used for the spin Hamiltonian simulations of the ferric low-spin species of cytochrome P450cam(solid lines in Figs.5 and 7).,Freeze-quench experiments I:,57,Fe(III)P450cam+substrate camphor+peroxy,acetic acid,The Mssbauer spectrum obtained at 4.2K and B=20 mT of cytochrome P450cam with the substrate camphor after reaction with peroxy acetic acid for 8 ms is shown in Fig.7.The spectrum displays an S=5/2-1/2 equilibrium which is characteristic for the substrate bound P450cam,but no Fe(IV)has been detected.The corresponding EPR spectrum(Fig.8)also shows an S=5/2-1/2 equilibrium.No characteristic signals for cpd I with S=1/2 or S