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ETp behavior. (a) Current density at -50 mV vs . inverse temperature of protium- and deuterium-labeled Az. (b) zoom-in on the high temperature part of (b) on a ln (J) scale. (c) Temperature dependence of the deuterium KIE. (d) A comparison between the effects of H/D substitution to the removal of Cu-ion (apo-Az).
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Measuring solid-state electron transport (ETp) across proteins allows studying electron transfer (ET) mechanism(s), while minimizing solvation effects on the process. ETp is, however, sensitive to any static (conformational) or dynamic (vibrational) changes in the protein. Our macroscopic measurements allow extending ETp studies to low temperatures...
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... secondary structure of Az is composed mainly from -sheet structure, which is supported by the hydrogen-bonding network of the amide backbone. Thus, substitution of the amide bond proton by deuterium should affect markedly the protein’s hydrogen bonding network (2, 31, 32). Consequently, this substitution should also affect the Cu coordination sphere of Az. The Cu coordination sphere is very sensitive to the hydrogen bonds in its vicinity, as shown by Marshall et al.,(33) who altered the hydrogen bonds in the second coordination sphere of the Cu site, causing marked changes in the protein’s reduction potential. In this context, Farver et al. (12) showed that the difference in the reduction potential between the deuterated- and protonated-Az is also temperature- dependent, with increasing difference with decreasing temperature. This difference in the reduction potential may well express a difference in flexibility of the protein, mainly around the Cu site (34-36). Changes in the protein flexibility have two outcomes (37): a) a structural aspect, i.e., as the protein is less flexible (equivalent to stiffer bonding) the reorganization energy for ET will be larger; b) a dynamic aspect, i.e., a less flexible protein has a shallower energy landscape as function of the (ET) reaction coordinate, which changes the ET activation energy. As a consequence, the difference in ETp as a function of temperature between protonated- and deuterated-Az (Fig. 3) may be a result of a change in the protein’s flexibility, induced by the change in the H(D)-bonds vibrational modes at the Cu coordination sphere. Although we do not know which vibrational modes are most affected by the H/D exchange, we can speculate that a more flexible protein (i.e., the deuterated-Az) will have lower energy frequency modes, , in comparison to a more rigid protein (the protonated-Az), as shown both experimentally (38) and theoretically (39) in other types of proteins. Thus, in the high-temperature regime of the deuterated Az sample, the lower energy frequency modes may cause a ...
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Citations
... 90 Nevertheless, a number of recent reports have demonstrated a substantial effect of deuteration on the rate of protein electron transfer. 17,41,91 This kinetic isotope effect can be attributed to altering dynamics of the protein upon deuteration for reactions controlled by dynamics (g > 1 in eq 9). 92 The existence of an unexpectedly strong kinetic isotope effect is an indication that these reactions fall into the regime of dynamical control. ...
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