Site-Directed Mutagenesis of Proline 52 To Glycine in Amicyanin Converts a True Electron Transfer Reaction into One that Is Conformationally Gated

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• 作者：John K. Ma ; Christopher J. Carrell ; F. Scott Mathews ; and Victor L. Davidson
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：July 11, 2006
• 年：2006
• 卷：45
• 期：27
• 页码：8284 - 8293
• 全文大小：197K
• 年卷期：v.45,no.27(July 11, 2006)
• ISSN：1520-4995

文摘

Amicyanin is a type I copper protein that is the natural electron acceptor for the quinoproteinmethylamine dehydrogenase (MADH). The conversion of Proline52 of amicyanin to a glycine does notalter the physical and spectroscopic properties of the copper binding site, but it does alter the rate ofelectron transfer (ET) from MADH. The values of electronic coupling (H_AB) and reorganization energy(mages/gifchars/lambda.gif" BORDER=0 >) that are associated with the true ET reaction from the reduced O-quinol tryptophan tryptophylquinone(TTQ) of MADH to oxidized amicyanin are significantly altered as a consequence of the P52G mutation.The experimentally determined H_AB increases from 12 to 78 cm^-1, and mages/gifchars/lambda.gif" BORDER=0 > increases from 2.3 to 2.8 eV.The rate and salt-dependence of the proton transfer-gated ET reaction from N-quinol MADH to amicyaninare also changed by the P52G mutation. Kinetic data suggests that a new common reaction step hasbecome rate-limiting for both the true and gated ET reactions that occur from different redox forms ofMADH. A comparison of the crystal structures of P52G amicyanin with those of native amicyanin freeand in complex with MADH provided clues as to the basis for the change in ET parameters. The mutationresults in the loss of three carbons from Pro52 and the movement of the neighboring residue Met51. Thisreduces the number of hydrophobic interactions with MADH in the complex and perturbs the protein-protein interface. A model is proposed for the ET reaction with P52G amicyanin in which the most stableconformation of the protein-protein complex with MADH is not optimal for ET. A new preceding kineticstep is introduced prior to true ET that requires P52G amicyanin to switch from this redox-inactive stablecomplex to a redox-active unstable complex. Thus, the ET reaction of P52G amicyanin is no longer atrue ET but one that is conformationally gated by the reorientation of the proteins within the ET proteincomplex. This same reaction step now also gates the ET from N-quinol MADH, which is normally rate-limited by a proton transfer.