1YQP

T268N mutant cytochrome domain of flavocytochrome P450 BM3

Summary for 1YQP

• Entry DOI: 10.2210/pdb1yqp/pdb
• Related: 1JME 1P0V 1P0W 1P0X 1YQO 2HPD
• Descriptor: Bifunctional P-450:NADPH-P450 reductase, PROTOPORPHYRIN IX CONTAINING FE (3 entities in total)
• Functional Keywords: cytochrome p450, fatty acid hydroxylase, oxidoreductase
• Biological source: Bacillus megaterium
• Cellular location: Cytoplasm (By similarity): P14779
• Total number of polymer chains: 2
• Total formula weight: 105597.90

Authors

Clark, J.P.,Miles, C.S.,Mowat, C.G.,Walkinshaw, M.D.,Reid, G.A.,Daff, S.N.,Chapman, S.K. (deposition date: 2005-02-02, release date: 2006-01-31, Last modification date: 2023-10-25)

Primary citation

Clark, J.P.,Miles, C.S.,Mowat, C.G.,Walkinshaw, M.D.,Reid, G.A.,Daff, S.N.,Chapman, S.K.
The role of Thr268 and Phe393 in cytochrome P450 BM3.
J.Inorg.Biochem., 100:1075-1090, 2006

PubMed Abstract: In flavocytochrome P450 BM3 there are several active site residues that are highly conserved throughout the P450 superfamily. Of these, a phenylalanine (Phe393) has been shown to modulate heme reduction potential through interactions with the implicitly conserved heme-ligand cysteine. In addition, a distal threonine (Thr268) has been implicated in a variety of roles including proton donation, oxygen activation and substrate recognition. Substrate binding in P450 BM3 causes a shift in the spin state from low- to high-spin. This change in spin-state is accompanied by a positive shift in the reduction potential (DeltaE(m) [WT+arachidonate (120 microM)]=+138 mV). Substitution of Thr268 by an alanine or asparagine residue causes a significant decrease in the ability of the enzyme to generate the high-spin complex via substrate binding and consequently leads to a decrease in the substrate-induced potential shift (DeltaE(m) [T268A+arachidonate (120 microM)]=+73 mV, DeltaE(m) [T268N+arachidonate (120 microM)]=+9 mV). Rate constants for the first electron transfer and for oxy-ferrous decay were measured by pre-steady-state stopped-flow kinetics and found to be almost entirely dependant on the heme reduction potential. More positive reduction potentials lead to enhanced rate constants for heme reduction and more stable oxy-ferrous species. In addition, substitutions of the threonine lead to an increase in the production of hydrogen peroxide in preference to hydroxylated product. These results suggest an important role for this active site threonine in substrate recognition and in maintaining an efficiently functioning enzyme. However, the dependence of the rate constants for oxy-ferrous decay on reduction potential raises some questions as to the importance of Thr268 in iron-oxo stabilisation.

PubMed: 16403573
DOI: 10.1016/j.jinorgbio.2005.11.020

Experimental method

X-RAY DIFFRACTION (1.8 Å)