Heme reduction, absorbance changes

The intramolecular electron transfer kg, subsequent to the rapid reduction, must occur because the Ru(III)-Fe(II) pairing is the stable one. It is easily monitored using absorbance changes which occur with reduction at the Fe(III) heme center. Both laser-produced Ru(bpy)3 and radicals such as CO (from pulse radiolysis (Prob. 15)) are very effective one-electron reductants for this task (Sec. 3.5).In another approach," the Fe in a heme protein is replaced by Zn. The resultant Zn porphyrin (ZnP) can be electronically excited to a triplet state, ZnP which is relatively long-lived (x = 15 ms) and is a good reducing agent E° = —0.62 V). Its decay via the usual pathways (compare (1.32)) is accelerated by electron transfer to another metal (natural or artificial) site in the protein e. g.. 

The very fast change relates to direct reduction of the Fe(III) center by the radical. The amount of absorbance change of this compared to the slow change can be understood if the hydrophobic nature of the heme site is considered. The rate of the slow change is similar for all systems since it involves a (common) intramolecular electron transfer. See (5.8.4). 

What are the electron transfer processes that occur after the initial flavin reduction In 1975, Capeillere-Blandin et al. (65) carried out combined stopped-flow and EPR rapid-freezing experiments on cleaved flavocytochrome 62 Simulation studies were used to derive an electron transfer scheme that would correlate with all the data (123). The time courses for FMN and heme reduction, which were biphasic in nature, were superimposable. The duration of phase 1 was 30-35 msec and accounted for approximately 85% of the total absorbance change observed. Phase 2 accounted for the remaining 15% and was some 20-fold slower than phase 1 (65). From EPR it was found that at the end of phase 1 up to 80% of the heme was reduced and up to 50% of the FMN... 

When similar reactions were carried out with Lb02, the absorbance changes were much slower. Early studies with mammalian heme proteins suggested that such reactions might give rise to free hydroxyl radicals via one-electron reduction of the hydrogen peroxide (100), but... 

Fig. 14. Effects of temperature on the absorbance of hemopexin and the N-domain of hemopexin. The unfolding of hemopexin and N-domain in 25 mM sodium phosphate, pH 7.4, was examined using absorbance spectroscopy (N. Shipulina et al., unpublished). The second derivative UV absorbance spectra of the protein moieties were used to follow protein unfolding and the Soret and visible region spectra to monitor the integrity of the heme complexes, as done with cytochrome 6502 (166). The ferri-heme complex is more stable than the apo-protein moiety, but the is slightly lower than that assessed by DSC, indicating that changes in conformation occur before thermodynamic unfolding. Reduction causes a large decrease in heme-complex stabihty, which is proposed to be a major factor in heme release from hemopexin by its cell membrane receptor, and addition of 150 mM sodium chloride enhanced the stabihty of ah forms of hemopexin.

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