Electron transfer in chemically and genetically modified myoglobins

The temperature dependences of the reduction potentials (Eo') of wildtype human myoglobin (Mb) and three site-directed mutants have been measured by using thin-layer spectroelectrochemistry. Residue Val68, which is in van der Waals contact with the heme in Mb, has been replaced by Glu, Asp, and Asn. At pH 7.0, reduction of the heme iron (III) in the former two proteins is accompanied by uptake of a proton by the protein. The changes in Eo', and the standard entropy (ΔSo') and enthalpy (ΔHo') of reduction in the mutant proteins were determined relative to values for wild-type; the change in Eo' at 25°C was about -200 millivolts for the Glu and Asp mutants, and about -80 millivolts for the Asn mutant. Reduction of Fe(III) to Fe(II) in the Glu and Asp mutants is accompanied by uptake of a proton. These studies demonstrate that Mb can tolerate substitution of a buried hydrophobic group by potentially charged and polar residues, and that such amino acid replacements can lead to substantial changes in the redox thermodynamics of the protein. Through analysis of the temperature dependence and shapes of NMR dispersion signals, it is determined that a water molecule is bound to the sixth coordination site of the ferric heme in the Val68Asp and in the Val68Asn recombinant proteins while the carboxyl group of the sidechain of Glu68 occupies this position in Val68Glu. The relative rhombic distortions in the ESR spectra of these mutant proteins combined with H217O and spin interconversion experiments performed on them confirm the conclusions of the NMRD study. The rates of intramolecular electron transfer (ET) of (NH3)5Ru-His48 (Val68Asp, His81GIn, Cys110AIa)Mb and (NH3)5Ru-His48 (Val68GIu,His81GIn,Cys110Ala)Mb were measured to be .85(3)s-1 and .30(2)s-1, respectively. This data supports the hypothesis that entropy of 111 reduction and reorganization energy of ET are inversely related. The rates of forward and reverse ET for (NH3)5 Ru-His48 (Val68GIu, His81 GIn, Cys110AIa)ZnMb -7.2(5)•104s-1and 1.4(2)•105s-1, respectively- demonstrate that the placement of a highly polar residue nearby does not significantly change the reorganization energy of the photoactive Zn porphyrin. The distal histidine imidazoles of (NH3)4isnRu-His48 SWMb and (NH3)5Ru-His48 SWMb were cyanated with BrCN. The intramolecular ET rates of these BrCN-modified Mb derivatives are 5.5(6)s-1 and 3.2(5)s-1, respectively. These respective rates are 20 and 10 times faster than those of their noncyanated counterparts after the differences in ET rate from driving force are scaled according to the Marcus equation. This increase in ET rate of the cyanated Mb derivatives is attributed to lower reorganization energy since the cyanated Mb heme is pentacoordinate in both oxidation states; whereas, the native Mb heme loses a water molecule upon reduction so that it changes from six to five coordinate. The reorganization energy from Fe-OH2 dissociation is estimated to be .2eV. This conclusion is used to reconcile data from previous experiments in our lab. ET in photoactive porphyrin-substituted myoglobins proceed faster than predicted by Marcus Theory when it is assumed that the only difference in ET parameters between photoactive porphyrins and native heme systems is driving force. However, the data can be consistently fit to Marcus Theory if one corrects for the smaller reorganization in the photoactive porphyrin systems since they do not undergo a coordination change upon ET. Finally, the intramolecular ET rate of (NH3)4isnRu-His48 SWMb was measured to be 3.0(4)s-1. This rate is within experimental error of that for (NH3)4pyrRu-His48 SWMb even though the former has 80mV more driving force. One likely possibility for this observation is that the tetraamminepyridineruthenium group undergoes less reorganization upon ET than the tetraammineisonicotinamideruthenium group. Moreover, analysis of the (NH3)4isnRu-His48 SWMb experimental system gives a likely explanation of why ET was not observed previously in (NH3)4isnRu-Cytochrome C.