Use of XAS for the elucidation of metal structure and function: applications to nickel biochemistry, molecular toxicology, and carcinogenesis.

Nickel has been shown to be an essential trace element involved in the metabolism of several species of bacteria, archea, and plants. In these organisms, nickel is involved in enzymes that catalyze both non-redox (e.g., urease, glyoxalase I) and redox (e.g., hydrogenase, carbon monoxide dehydrogenase, superoxide dismutase) reactions, and proteins involved in the transport, storage, metallocenter assembly, and regulation of nickel concentration have evolved. Studies of structure/function relationships in nickel biochemistry reveal that cysteine ligands are used to stabilize the Ni(III/II) redox couple. Certain nickel compounds have also been shown to be potent human carcinogens. A likely target for carcinogenic nickel is nuclear histone proteins. Here we present X-ray absorption spectroscopic studies of a model Ni peptide designed to help characterize the structure of the nickel complexes formed with histones and place them in the context of nickel structure/function relationships, to gain insights into the molecular mechanism of nickel carcinogenesis

Impact of BRAF, MLH1 on the incidence of microsatellite instability high colorectal cancer in populations based study

We have identified an alternative pathway of tumorigenesis in sporadic colon cancer, involving microsatellite instability due to mismatched repair methylation, which may be driven by mutations in the BRAF gene (V600E). Colorectal cancer (CRC) is the most common cancer in the world, and African Americans show a higher incidence than other populations in the United States. We analyzed sporadic CRCs in Omani (of African origin, N = 61), Iranian (of Caucasian origin, N = 53) and African American (N = 95) patients for microsatellite instability, expression status of mismatched repair genes (hMLH1, hMSH2) and presence of the BRAF (V600E) mutation. In the Omani group, all tumors with BRAF mutations were located in the left side of the colon, and for African Americans, 88% [7] of tumors with BRAF mutations were found in the right side of the colon. In African Americans, 31% of tumors displayed microsatellite instability at two or more markers (MSI-H), while this rate was 26% and 13% for tumors in the Iranian and Omani groups, respectively. A majority of these MSI-H tumors were located in the proximal colon (right side) in African American and Iranian subjects, whereas most were located in the distal colon (left side) in Omani subjects. Defects in hMLH1 gene expression were found in 77% of MSI-H tumors in both African Americans and Iranians and in 38% of tumors in Omanis. BRAF mutations were observed in all subjects: 10% of tumors in African Americans (8/82), 2% of tumors in Iranians (1/53), and 19% of tumors in Omanis (11/59). Our findings suggest that CRC occurs at a younger age in Omani and Iranian patients, and these groups showed a lower occurrence of MSI-H than did African American patients. Our multivariate model suggests an important and significant role of hMLH1 expression and BRAF mutation in MSI-H CRC in these populations. The high occurrence of MSI-H tumors in African Americans may have significant implications for treatment, since patients with MSI-H lesions display a different response to chemotherapeutic agents such as 5-fluorouracil

Structural investigations of nickel metalloproteins: The Nik system (NikA and NikR), acireductone dioxygenase, and hydrogenase

Nickel, essential to many biological processes, is a component of the active sites of enzymes that catalyze both redox and non-redox reactions. Here the results of investigations aimed at understanding the structure and function of four nickel metalloproteins is presented. X-ray absorption spectroscopy (XAS) was used to explore the nickel sites in Escherichia coli NikA and NikR. NikA is a Ni-binding component of the NikA-E nickel specific permease, responsible for the uptake of Ni in E. coli. NikA also plays a role in the negative chemotaxis associated with toxic Ni concentrations. NikA plays a non-redox role, and XAS of its nickel site exhibited an octahedral environment composed of six N/O-ligands. NikR regulates Ni uptake and translocation by regulation of the nik operon. XAS of the high affinity nickel sites of NikR holoprotein, C-terminal domain, and a half-site DNA complex revealed a planar four coordinate, a five coordinate, and an octahedral site respectively. There is a predominance of N/O-ligation for the NikR samples as expected for Ni proteins with a non-redox role. Only the holoprotein possesses one sulfur ligand. The Ni coordination environments support a proposed role for NikR function where the N-terminus binds DNA specific subsites, and the C-terminal senses, and binds nickel and stabilizes the NikR-DNA complex. XAS was used to probe the structure of the novel nickel containing acireductone dioxygenase (NiARD). Klebsiella pneumoniae produces two ARD\u27s, involved in its methionine salvage pathway. They differ only in their metal content, but catalyze different reactions. NiARD provides a shunt to the methionine salvage pathway. XAS of resting NiARD and the enzyme substrate complex shows an octahedral resting NiARD with N/O-ligands, where 3 or 4 are histidines. Substrate binds to NiARD in a bidentate fashion displacing two ligands, at least one being a histidine. Nickel also plays a key redox role in NiFe hydrogenases. Here circular dichroism and electronic spectroscopy were used to monitor conformational changes in Thiocapsa roseopersicina NiFe hydrogenase as a function of redox poise. Although changes may occur at the metal sites, the resulting changes in the protein secondary structure are small

XAS investigation of the structure and function of Ni in acireductone dioxygenase

6761-676

Use of XAS for the elucidation of metal structure and function: Applications to nickel biochemistry, molecular toxicology, and carcinogenesis

705-70

Regulation of ni uptake in E-coli.

U156-U15