Journal
PROTEIN SCIENCE
Volume 14, Issue 9, Pages 2414-2420Publisher
WILEY
DOI: 10.1110/ps.051459705
Keywords
X-ray crystallography; thioredoxin; disulfide; peroxiredoxin; thiolate; alkyl hydroperoxide reductase; synchrotron radiation; pK(a); radiation damage
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Funding
- NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES [P30ES000210, P01ES000040] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM050389] Funding Source: NIH RePORTER
- NIEHS NIH HHS [P30 ES00210, P01 ES000040, ES00040, P30 ES000210] Funding Source: Medline
- NIGMS NIH HHS [R01 GM050389, R01 GM50389, R01 GM050389-12] Funding Source: Medline
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The flavoprotein component (AhpF) of Salmonella typhimurium alkyl hydroperoxide reductase contains an N-terminal domain (NTD) with two contiguous thioredoxin folds but only one redox-active disulfide (within the sequence -Cys(129)-His-Asn-Cys(132)-). This active site is responsible for mediating the transfer of electrons from the thioredoxin reductase-like segment of AhPF to AhpC, the peroxiredoxin component of the two-protein peroxidase system. The previously reported crystal structure of AhpF possessed a reduced NTD active site, although fully oxidized protein was used for crystallization. To further investigate this active site, we crystallized an isolated recombinant NTD (rNTD); using diffraction data sets collected first at our in-house X-ray source and subsequently at a synchrotron, we showed that the active site disulfide bond (Cys129-Cys132) is oxidized in the native crystals but becomes reduced during synchrotron data collection. The NTD disulfide bond is apparently particularly sensitive to radiation cleavage compared with other protein disulfides. The two data sets provide the first view of an oxidized (disulfide) form of NTD and show that the changes in conformation upon reduction of the disulfide are localized and small. Furthermore, we report the apparent pK(a) of the active site thiol to be similar to 5.1, a relatively low pK(a) given its redox potential (-265 mV) compared with most members of the thioredoxin family.
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