Electronic Structural Information from Q-Band ENDOR on the Type 1 and Type 2 Copper Liganding Environment in Wild-Type and Mutant Forms of Copper-Containing Nitrite Reductase

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• 作者：Andrei Veselov ; Kenneth Olesen ; Andrzej Sienkiewicz ; James P. Shapleigh ; and Charles P. Scholes
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：April 28, 1998
• 年：1998
• 卷：37
• 期：17
• 页码：6095 - 6105
• 全文大小：119K
• 年卷期：v.37,no.17(April 28, 1998)
• ISSN：1520-4995

文摘

Q-band ENDOR elucidated proton and nitrogen hyperfine features toprovide spin densityinformation at ligands of blue-green Type 1 and catalytic Type 2 coppercenters in nitrite reductase. Theblue-green Type 1 center of nitrite reductase has a redox,electron-transfer role, and compared to the bluecenter of plastocyanin, it has the following structural differences:a shortened Cu-S_met bond length, alonger Cu-S_cys bond length, and alteredligand-copper-ligand bond angles (Adman, E. T., Godden,J.W., and Turley, S. (1995) J. Biol. Chem. 270,27458-27474). The hyperfine couplings of the twoType1 histidine (N_{ages/gifchars/delta.gif" BORDER=0 >}) ligands showed a larger percentagedifference from each other in electron spin densitythan previously reported for other blue Type 1 proteins, while thecysteine ages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-proton hyperfine couplings,a measure of unpaired pages/gifchars/pi.gif" BORDER=0 > spin density on the liganding cysteinesulfur, showed a smaller electron spindensity. A mutation of the Type 1 center, M182T, having thecopper-liganding Met¹⁸² transformed toThr¹⁸², caused the center to revert to an optically"blue" center, raised its redox potential by ~100mV,and led to the loss of activity (prior paper). Surprisingly, inM182T there was no change from nativeType 1 copper either in the histidine or cysteine hyperfine couplingsor in g values and Cu nuclear hyperfinecouplings. The conclusion is that the optical and redoxalterations due to changed Type 1 methionineligation need not be concurrent with electron spin delocalizationchanges in the HOMO as reported fromits essential cysteine and histidines. A detailed picture of thenitrogen couplings from the three histidine(N_{ages/gifchars/epsilon.gif" BORDER=0 >}) ligands of the Type 2 center indicated a substantial(~200%) electronic hyperfine inequivalence ofone of the histidine nitrogens from the other two within the Type 2HOMO and thus provided evidencefor electronic distortion of the Type 2 site. In the presence ofthe nitrite substrate, hyperfine couplingsof all histidines diminished. We suggest that this nitrite-induceddecreased covalency would correlatewith an increased Type 2 redox potential to assist electron transfer tothe Type 2 center. Dipole-coupled,angle-selected exchangeable proton features, observed over a range ofg values, predicted a ligand-waterproton distance of 2.80 Å from copper, and these water protons wereeliminated by nitrite. His²⁸⁷ is nota Type 2 ligand but is positioned to perturb an axial water or anitrite of Type 2 copper. In the presenceof nitrite the mutant H287E showed no evidence for the loss of waterprotons and no diminished ligandhistidine covalency. H287E has vastly diminished activity (priorpaper), and the ENDOR information isthat NO₂^- does not bind to Type 2 copper ofH287E. In summary, the electronic information fromthisstudy of native and suitably chosen mutants provided a test of thehighest occupied molecular orbital(HOMO) wave function at Type 1 and Type 2 coppers and an intimateelectronic insight into functionalenzymatic properties.