Detection of heme oxygenase activity in a library of four-helix bundle proteins: towards the de novo synthesis of functional heme proteins.
Design and chemical synthesis of de novo heme proteins with enzymatic activity on cellulose membranes is described. 352 antiparallel four-helix bundle proteins with a single histidine for heme ligation were assembled from three different sets of short amphipathic helices on membrane-bound peptide templates. The templates were coupled by linkers to cellulose membranes of microplate format, which could be cleaved for control of intermediate and final products. The incorporation of heme and the heme oxygenase activity of the 352 proteins were monitored by measuring UV-visible spectra directly on the cellulose. The kinetics of the heme oxygenase reaction was studied by monitoring the decrease of the Soret band and the transient absorbance of verdoheme being an intermediate product in the formation of biliverdin. Four of the proteins covering a broad range of the enzymatic rate constants were selected and synthesized in solution for further characterization. Detailed studies by redox potentiometry, analytical ultracentrifugation, and electron paramagnetic resonance spectroscopy yielded information about the aggregation state of the proteins, the spin state and the putative coordination environment of the iron. The amount of five-coordinated high-spin iron and a positive reduction potential were found to promote the oxygenase activity of the proteins.
Monien
Bernhard H
BH
Institute of Biology II / Biochemistry, Albert-Ludwigs-Universität Freiburg, Schänzlestr. 1, D-79104 Freiburg, Germany. monien@dife.de
Drepper
Friedel
F
Sommerhalter
Monika
M
Lubitz
Wolfgang
W
Haehnel
Wolfgang
W
D000595:Amino Acid Sequence; D002384:Catalysis; D002482:Cellulose / Q000378:metabolism; D002498:Centrifugation; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism*; D006420:Hemeproteins / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011524:Protoporphyrins / Q000737:chemistry; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors; D017186:Titrimetry
D006420:Hemeproteins; D019151:Peptide Library; D010455:Peptides; D011524:Protoporphyrins; D012996:Solutions; D006418:Heme; D002482:Cellulose; C028025:protoporphyrin IX; D006419:Heme Oxygenase (Decyclizing)