Relations between optical spectrum and structure in nitrosyl hemoglobin and hybrids.

Abstract

The addition of inositolhexaphosphate to stripped nitrosyl hemoglobin provokes strong changes in the visible absorption spectrum of the protein. The stoichiometry of this reaction, determined by spectrophotometry, is one mole of IHP per tetramer. The equilibrium dissociation constant of IHP for Hb NO , in 0·1 m -2,2-bis (hydroxymethyl)-2,2′,2″-nitrilotriethanol (pH 6·8) is 1·3×10 −6 m and increases with ionic strength and pH. IHP has only a small effect on bis ( N -maleimidomethyl) ether hemoglobin. The spectral changes induced by IHP on hemoglobin nitrosyl hybrids α 2 CO β 2 NO and α 2 NO β 2 CO have been determined. Only the latter exhibits large changes in absorbance. Comparison between the Δ∈ values found for Hb NO and α 2 NO β 2 CO suggests that in Hb NO , the α NO subunit alone contributes to the spectral changes. IHP modifies also the ultraviolet absorption spectrum of Hb NO , and decreases the reactivity of cysteine β93 toward p -chloromercurybenzoate. These changes are qualitatively similar to those that appear on deoxygenation of oxyhemoglobin, and on binding IHP to aquomethemoglobin, as previously documented by Perutz et al. (1974 a ) . The suggestion that IHP induces a new quaternary conformation in Hb NO with deoxy character is also supported by the finding that spectral changes exactly opposite to those described in the reaction of IHP with Hb NO are induced in the absence of IHP after fixation of a ligand to α 2 NO β 2 deoxy hybrid, a reaction that probably consists of a change in the structure of this hybrid from a deoxy to an oxy conformation. The results are discussed in terms of the stereochemical hypothesis suggested by Perutz et al. (1974 b ) , according to which the allosteric equilibrium constant L in hemoglobin is governed by the Fe−N 8His distance. Kinetic studies have shown that the optical changes in Hb NO and α 2 NO β 2 deoxy hybrid are slow and complex, involving Hb NO dimers and tetramers. First and indirect estimates of the tetramer-dimer equilibrium constant of Hb NO are given. They are K 4/2 =4·2×10 −6 m and 20×10 −6 m , respectively, in 0·1 m -phosphate and 0·05 m -2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol buffers (pH 6·8). It is also demonstrated that the IHP-induced spectral changes in Hb NO owe their origin to a slow rearrangement of the α NO subunit inside a quaternary structure which has already changed in conformation, as suggested by the criterion of cysteine β93 reactivity. Evidence is provided that artificially prepared α 2 NO β 2 deoxy hybrid differs some-what in conformation from naturally occurring hemoglobin intermediates, as was suggested already for valency hybrids by Cassoly & Gibson (1972) .