Spectroscopic Evidence of the Role of an Axial Ligand Histidinate in the Mechanism of Adrenal Cytochrome b561
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- 作者：Giordano F. Z. da Silva ; Vladimir P. Shinkarev ; Yury A. Kamensky ; Graham Palmer
- 刊名：Biochemistry
- 出版年：2012
- 出版时间：November 6, 2012
- 年：2012
- 卷：51
- 期：44
- 页码：8730-8742
- 全文大小：608K
- 年卷期：v.51,no.44(November 6, 2012)
- ISSN：1520-4995
文摘
Adrenal cytochrome b561 (AdCytb) is the prototype of a widespread protein family that specializes in delivering electrons donated by ascorbic acid for different processes in eukaryotic cells. AdCytb transports redox equivalents from cytoplasmic ascorbate across the membranes of chromaffin granules to support norepinephrine synthesis within their matrix. The interaction of AdCytb with ascorbate is central to a proposed mechanism of AdCytb鈥檚 function, and a histidine in the active site of AdCytb was suggested to bind cytoplasmic ascorbate and serve as the acceptor of the proton released during ascorbate oxidation. AdCytb contains high- and low-potential hemes but their orientation relative to the matrix and cytoplasmic interfaces of chromaffin granule membrane is disputed. Using a combination of three spectroscopic methods (UV鈥搗is absorption, near-infrared magnetic circular dichroism, and electron paramagnetic resonance), we find that a histidine residue that serves as an axial ligand to the high-potential heme undergoes deprotonation with a pK of 8.0 and is thus a good candidate for interaction with cytoplasmic ascorbate. The low-potential heme of AdCytb is found to have a pK of 10.5, making it an unlikely candidate for accepting a proton at physiological pH. UV鈥搗is spectroscopy reveals an additional proton acceptor group in AdCytb with a pK of 6.5 that is not observed by the other two techniques; whether it plays a role in the mechanism of AdCytb is unknown. We incorporate these results into an updated mechanism of AdCytb reduction predicated on the high-potential heme鈥檚 localization on the cytoplasmic interface of the chromaffin granule membrane.