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These changes have been observed spectroscopically and are also accompanied by a lowering of the heme reduction potential of the single mutant (177 mV vs SHE) relative to that of wt (236 mV).
The combination of an increased degree of ruffling and a stronger Fe(III)–His interaction is suggested to be the cause for a decrease in reduction potential for the double mutant (155 mV vs SHE) relative to that of wt (236 mV).
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