Function of the [2FE-2S] cluster in mammalian ferrochelatase: a possible role as a nitric oxide sensor.
Ferrochelatase (E.C. 4.99.1.1) is the terminal enzyme of the heme biosynthetic pathway, catalyzing the insertion of ferrous iron into protoporphyrin. In mammals the enzyme contains a labile [2Fe-2S] center. Although this cluster is absent in all prokaryotic, plant, and yeast ferrochelatases, its destruction or elimination from the mammalian enzyme results in loss of enzyme activity. In the current study we present data which clearly demonstrate that mammalian ferrochelatase is strongly inhibited by nitric oxide and that this effect is mediated via destruction of the [2Fe-2S] cluster. Carbon monoxide has no inhibitory effect, and yeast ferrochelatase, which lacks the [2Fe-2S] cluster, is not affected by NO (or CO). EPR and UV-visible absorption of purified recombinant human ferrochelatase provides evidence that NO is targeting the [2Fe-2S] center. UV-visible absorption spectroscopy of both human and murine recombinant ferrochelatase incubated with NO or the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), indicate a rapid loss of the visible absorption spectrum of the [2Fe-2S] cluster. EPR studies of the resulting samples reveal the characteristic axial S = 1/2 resonance, g perpendicular = 2.033, and g parallel = 2.014 of a cysteinyl-coordinated monomeric iron-dinitrosyl cluster degradation product. Parallel spectroscopic studies of spinach ferredoxin, which also contains a [2Fe-2S] cluster, gave no indication of NO-induced cluster degradation under the same experimental conditions. Exposure of DMSO-induced murine erythroleukemia cells exposed to SNAP results in an initial decrease in heme production, suggesting that in vivo the cluster is rapidly destroyed. The potential physiological relevance of these data to the anemias that are found in individuals with chronic infections is discussed.
Sellers
V M
VM
Department of Microbiology, University of Georgia, Athens, Georgia 30602, USA.
Johnson
M K
MK
Dailey
H A
HA
D000818:Animals; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D005294:Ferrochelatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000096:biosynthesis; D006801:Humans; D066298:In Vitro Techniques; D007501:Iron / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013455:Sulfur / Q000737:chemistry; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D009569:Nitric Oxide; D006418:Heme; D013455:Sulfur; D007501:Iron; D005294:Ferrochelatase