Heme Binding Properties
of Glyceraldehyde-3-phosphate
Dehydrogenase
- Publication date
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Abstract
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a
glycolytic
enzyme that also functions in transcriptional regulation, oxidative
stress, vesicular trafficking, and apoptosis. Because GAPDH is required
for the insertion of cellular heme into inducible nitric oxide synthase
[Chakravarti, R., et al. (2010) <i>Proc. Natl. Acad. Sci. U.S.A.
107</i>, 18004–18009], we extensively characterized the
heme binding properties of GAPDH. Substoichiometric amounts of ferric
heme bound to GAPDH (one heme per GAPDH tetramer) to form a low-spin
complex with UV–visible maxima at 362, 418, and 537 nm and
when reduced to ferrous gave maxima at 424, 527, and 559 nm. Ferric
heme association and dissociation rate constants at 10 °C were
as follows: <i>k</i><sub>on</sub> = 17800 M<sup>–1</sup> s<sup>–1</sup>, <i>k</i><sub>off1</sub> = 7.0 ×
10<sup>–3</sup> s<sup>–1</sup>, and <i>k</i><sub>off2</sub> = 3.3 × 10<sup>–4</sup> s<sup>–1</sup> (giving approximate affinities of 19–390 nM). Ferrous heme
bound more poorly to GAPDH and dissociated with a <i>k</i><sub>off</sub> of 4.2 × 10<sup>–3</sup> s<sup>–1</sup>. Magnetic circular dichroism, resonance Raman, and electron paramagnetic
resonance spectroscopic data on the ferric, ferrous, and ferrous–CO
complexes of GAPDH showed that the heme is bis-ligated with His as
the proximal ligand. The distal ligand in the ferric complex was not
displaced by CN<sup>–</sup> or N<sub>3</sub><sup>–</sup> but in the ferrous complex could be displaced by CO at a rate of
1.75 s<sup>–1</sup> (for >0.2 mM CO). Studies with heme
analogues
revealed selectivity toward the coordinating metal and porphyrin ring
structure. The GAPDH–heme complex was isolated from bacteria
induced to express rabbit GAPDH in the presence of δ-aminolevulinic
acid. Our finding of heme binding to GAPDH expands the protein’s
potential roles. The strength, selectivity, reversibility, and redox
sensitivity of heme binding to GAPDH are consistent with it performing
heme sensing or heme chaperone-like functions in cells