Radical S-adenosylmethionine enzyme coproporphyrinogen III oxidase HemN: functional features of the [4Fe-4S] cluster and the two bound S-adenosyl-L-methionines.
The S-adenosylmethionine (AdoMet) radical enzyme oxygen-independent coproporphyrinogen III oxidase HemN catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX during bacterial heme biosynthesis. The recently solved crystal structure of Escherichia coli HemN revealed the presence of an unusually coordinated iron-sulfur cluster and two molecules of AdoMet. EPR spectroscopy of the reduced iron-sulfur center in anaerobically purified HemN in the absence of AdoMet has revealed a [4Fe-4S](1+) cluster in two slightly different conformations. Mössbauer spectroscopy of anaerobically purified HemN has identified a predominantly [4Fe-4S](2+) cluster in which only three iron atoms were coordinated by cysteine residues (isomer shift of delta = 0.43 (1) mm/s). The fourth non-cysteine-ligated iron exhibited a delta = 0.57 (3) mm/s, which shifted to a delta = 0.68 (3) mm/s upon addition of AdoMet. Substrate binding by HemN did not alter AdoMet coordination to the cluster. Multiple rounds of AdoMet cleavage with the formation of the reaction product methionine indicated AdoMet consumption during catalysis and identified AdoMet as a co-substrate for HemN catalysis. AdoMet cleavage was found to be dependent on the presence of the substrate coproporphyrinogen III. Two molecules of AdoMet were cleaved during one catalytic cycle for the formation of one molecule of protoporphyrinogen IX. Finally, the binding site for the unusual second, non iron-sulfur cluster coordinating AdoMet molecule (AdoMet2) was targeted using site-directed mutagenesis. All AdoMet2 binding site mutants still contained an iron-sulfur cluster and most still exhibited AdoMet cleavage, albeit reduced compared with the wild-type enzyme. However, all mutants lost their overall catalytic ability indicating a functional role for AdoMet2 in HemN catalysis. The reported significant correlation of structural and functional biophysical and biochemical data identifies HemN as a useful model system for the elucidation of general AdoMet radical enzyme features.
Layer
Gunhild
G
Institut für Mikrobiologie, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany.
Grage
Katrin
K
Teschner
Thomas
T
Schünemann
Volker
V
Breckau
Daniela
D
Masoumi
Ava
A
Jahn
Martina
M
Heathcote
Peter
P
Trautwein
Alfred X
AX
Jahn
Dieter
D
D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D001665:Binding Sites; D002384:Catalysis; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003304:Coproporphyrinogen Oxidase / Q000378:metabolism / Q000502:physiology*; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D005609:Free Radicals; D007501:Iron / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry; D011485:Protein Binding; D050768:Protoporphyrinogen Oxidase; D011994:Recombinant Proteins / Q000737:chemistry; D012436:S-Adenosylmethionine / Q000737:chemistry*; D013053:Spectrophotometry; D015204:Spectroscopy, Mossbauer; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013997:Time Factors
D001426:Bacterial Proteins; D003304:Coproporphyrinogen Oxidase; D003545:Cysteine; D005609:Free Radicals; D007501:Iron; D007506:Iron-Sulfur Proteins; D008024:Ligands; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; C109194:HemN protein, Bacteria