Effect of the disease-causing R266K mutation on the heme and PLP environments of human cystathionine β-synthase.
Cystathionine β-synthase (CBS) is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme of the transsulfuration pathway that condenses serine with homocysteine to form cystathionine; intriguingly, human CBS also contains a heme b cofactor of unknown function. Herein we describe the enzymatic and spectroscopic properties of a disease-associated R266K hCBS variant, which has an altered hydrogen-bonding environment. The R266K hCBS contains a low-spin, six-coordinate Fe(III) heme bearing a His/Cys ligation motif, like that of WT hCBS; however, there is a geometric distortion that exists at the R266K heme. Using rR spectroscopy, we show that the Fe(III)-Cys(thiolate) bond is longer and weaker in R266K, as evidenced by an 8 cm(-1) downshift in the ν(Fe-S) resonance. Presence of this longer and weaker Fe(III)-Cys(thiolate) bond is correlated with alteration of the fluorescence spectrum of the active PLP ketoenamine tautomer. Activity data demonstrate that, relative to WT, the R266K variant is more impaired in the alternative cysteine-synthesis reaction than in the canonical cystathionine-synthesis reaction. This diminished cysteine synthesis activity and a greater sensitivity to exogenous PLP correlate with the change in PLP environment. Fe-S(Cys) bond weakening causes a nearly 300-fold increase in the rate of ligand switching upon reduction of the R266K heme. Combined, these data demonstrate cross talk between the heme and PLP active sites, consistent with previous proposals, revealing that alteration of the Arg(266)-Cys(52) interaction affects PLP-dependent activity and dramatically destabilizes the ferrous thiolate-ligated heme complex, underscoring the importance of this hydrogen-bonding residue pair.
Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706, USA.
Aaron T
AT
Smith
Yang
Y
Su
Daniel J
DJ
Stevens
Tomas
T
Majtan
Jan P
JP
Kraus
Judith N
JN
Burstyn
D020134:Catalytic Domain; D002942:Circular Dichroism; D003541:Cystathionine beta-Synthase / Q000737:chemistry* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000235:genetics*; D006712:Homocystinuria / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013059:Spectrum Analysis, Raman; D013696:Temperature
D005296:Ferrous Compounds; D006418:Heme; D011732:Pyridoxal Phosphate; D003541:Cystathionine beta-Synthase