Hyperfine-shifted proton resonances ferrous

Fig. 25. 250-MHz H NMR spectra of Hb A in both deoxy and oxy forms in 0.1 M Bis-Tris plus 10 mM IHP in D20 at pH 6.6 at 27°C. The resonance at 27 ppm was that of the NMR shift reagent. This reference signal was used for the intensity calibration of the ferrous hyperfine-shifted proton resonances. [From Ho et al. (1982b)].

Fig. 39. 90-MHz H NMR spectra of ferrous hyperfine-shifted proton resonances of deoxyhemoglobins containing mutations in the a,(32 subunit interface in 0.1 Af phosphate in D20 at pH 6.6 and 25°C. [Adapted from Davis et al. (1971) and Ho and Russu (1985)].

Fig. 41. Variation of the ferrous hyperfine-shifted proton resonances of Hb A in 0.1 M Bis-Tris plus 10 mM IHP as a function of oxygenation at pH 6.6 and 27°C A, a-heme resonance at +12 ppm from HDO (data from Viggiano et al., 1979) , a-heme resonance at + 12 ppm from HDO (data from Ho et al., 1982b) , a-heme resonance at + 12 ppm from H20 (data from Viggiano and Ho, 1979) o, p-heme resonance at +18 ppm from HDO (data from Viggiano et al., 1979) , p-heme resonance at + 18 ppm from HDO (data from Hoetal., 1982b) x, p-heme resonance at + 16.9 ppm from HDO (data from Ho et al., 1982b) the curve is the fraction of fully deoxy-Hb tetramers calculated from the data of Tyuma et al. (1973). [From Ho et al. (1982b)].

First, comparing the ferrous hyperfine-shifted proton resonances... 

At high fields the NMR spectrum of cyanoferricytochrome c contains resolved hyperfine-shifted fines at +1.2 and +4.4 ppm (Fig. 29). Upon addition of dithionite these fines, as well as the resonances between —10 and —30 ppm, disappear immediately, indicating fast reduction to the diamagnetic ferrous oxidation state. The primary reaction product has no proton resonances at higher field than +1 ppm. However, with time elapsing after the reduction the NMR spectrum of ferrocytochrome c appears slowly. On the other hand the entire ferrocytochrome c spectrum was observed immediately after reduction of ferricytochrome c. 

Figures 49 and 50 show the 600-MHz H NMR spectra of cross-linked mixed-valency asymmetric hybrid Hbs with one cyanomet chain per Hb tetramer. Figures 49A and 50A give the proton resonances of (a+CNP)A(aP)cXL and (aP+CN)A(a(3)cXL in D20 from +7 to +20 ppm from HDO. These hyperfine-shifted resonances arise from the protons in the heme groups and/or the protons of the amino acid residues in the immediate surroundings of the heme group. They are shifted from their usual diamagnetic positions by hyperfine interactions between the unpaired electrons from the low-spin ferric and high-spin ferrous heme iron atoms and the nearby protons. As discussed earlier, they are very sensitive to the environment of the heme pocket. The signals from the subunits with high-spin ferrous heme iron are much broader than those from cyanomet (low-spin...

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