Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants

M Ikeda-Saito,H Hori,L.A Andersson,R.C Prince,I.J Pickering,G.N George,C R Sanders,R.S Lutz,E.J Mckelvey,R Mattera

doi:10.1016/s0021-9258(18)50024-7

M Ikeda-Saito, H Hori + Show 8 more

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https://doi.org/10.1016/s0021-9258(18)50024-7

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Abstract

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.

Highlights

From the $Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio
The and Glycera Hb (Giacometti et al, 1981; Mintorovitch and distal Val and distal Leu ferric myoglobin mutants at Satterlee, 1988) are significantly different, both in extincneutral pH exhibitedEPRspectrawith g, values tion and maxima, from those of typical ferric Mbs and smaller than 6, which could be interpreted as an ad- Hbs
The optical spectral properties of ferric Mb H64Q are similar to those of H64H, but differences are noticed between these two spectra; there is a shoulder on the blue side of the Soret band, anda 4-nm red shift of the socalled charge transfer band to 637 nm upon His + Gln mutation

Summary

Recombinanthuman myoglobin mutantswiththe

The coordination and electronic structures of heme iron distal His residue (E7, Hiss4)replaced by Leu, Val, or in ferric hemoproteins andheme modelsystems have been. Based on the spectroscopic properties of recombinant human Mb, Varadarajan et al (1989) concluded that the coordination structure of the heme group in ferric human recombinant Mb is essentially the same as that of sperm whale Mb. As shown below, our present MCD, NMR, and EPR datafurther support theirconclusions: ferric human Mb is a high spin complex with a water molecule as its sixth ligand at neutral pH, and its alkaline form is a low spin complex with an OH ligand, as established in the better characterized authentic and recombinant sperm whale Mb proteins (Antonini and Brunori, 1971; Blumberg and Peisach, 1971; Peisach et al, 1984; Morikis et al, 1990; Phillips et al, 1990)

RESULTS

Soret fa Visible

Ferric Mb

Protein n

DISCUSSION