Zinc and Magnesium Substitution in Hemoglobin: Cyclic Electron Transfer within Mixed-Metal Hybrids and Crystal Structure of MgHb

Studies of long-range electron transfer within mixed-metal hemoglobin (Hb) hybrids [α₂(FeP), β₂(MP)] (M = Mg, Zn; P = protoporphyrin IX) are reported, along with the X-ray crystal structure of magnesium-substituted hemoglobin (MgHb). MgHb adopts the quaternary structure of deoxyHb, and replacement of Fe by Mg causes negligible structural changes, supporting earlier inferences that electron transfer (ET) in these hybrids occurs between redox centers held at fixed and crystallographically known distance and orientation. Upon flash photolysis of the [MP, Fe³⁺(H₂O)P] hybrids, the charge-separated intermediate [(MP)⁺, Fe²⁺P] (I) is formed by a photoinitiated ³(MP) → Fe³⁺P intramolecular electron-transfer process with rate constant k_t, and returns to the ground state by a Fe²⁺P → (MP)⁺ thermal electron transfer with rate constant k_b. By use of the transient absorption technique, we have measured k_t and k_b for M = Mg and Zn as a function of temperature between 0 and 25 °C. The rate constant, k_t = 35 (8) s⁻¹ for Mg at room temperature is significantly lower than that of Zn, k_t = 85 (15) s⁻¹, although the driving force is greater in the former by about 100 mV. The charge recombination rate, k_b, within [Zn, Fe] is 350 (35) s⁻¹ compared to that of 155 (15) s⁻¹ for [Mg, Fe]. The inequality, k_b,t^Zn ≠ k_b,t^Mg, rules out rate-limiting conformational “gating” for photoinitiated and thermally activated electron-transfer processes, and further indicates that in both cases ET is direct. Comparisons among the rate constants indicate that the electronic-coupling matrix element between redox sites, |H_AB|², may be slightly (roughly 2-fold) greater for M = Zn than for M = Mg.