Isolation, structural determination, and characterization of a novel copper-binding compound from Methylosinus trichosporium OB3b

Methanotrophic bacteria play a significant role in the global methane budget by utilizing methane as their source of carbon and energy. In some methanotrophs, such as the Type II Methylosinus trichosporium OB3b, the enzyme responsible for methane oxidation, methane monooxygenase (MMO), can also rapidly cooxidize a number of low-molecular-weight halogenated pollutants such as trichloroethylene (TCE). These organisms thus possess tremendous potential for bioremediation processes; however, much of the progress in its biotechnical applications has been hampered by a lack of understanding of its copper-related physiology that significantly affects cooxidation rates. A key step in understanding this aspect of methanotroph physiology is the isolation and structural determination of a small copper-binding compound (CBC) from these organisms.; Copper Binding Compound (CBC) from Methylosinus trichosporium OB3b is a small yellow, fluorescent chromopeptide that is secreted into its growth medium when the cultures are starved for copper. The compound was isolated and purified to homogeneity using Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) together with Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) mass spectrometry to monitor each step of the purification process. Its tendency to breakdown during purification was overcome by stabilizing the compound with CUCl2 before purification. The molecular mass of the purified compound bound to copper was determined to be 1217.2 Da using MALDI-TOF MS. The mass of the corresponding metal-free compound was 62 mass units less. Investigation of its fluorescent properties indicated that the compound contained three fluorescent functions each exhibiting distinct emission and excitation.; The sequence of the molecule's amino acid backbone was determined using a combination of Edman degradation and peptide ladder sequencing using partial acid and proteolytic degradation with subsequent mass spectral (MS) analysis of the generated fragments. Tandem MS (MS/MS) was also utilized to obtain sequence information. The final structure of CBC was determined by growth of diffraction quality crystals using vapor diffusion and subsequent X-ray analysis. The 1.1 A crystal structure revealed that CBC was indeed a copper-containing chromopeptide comprising of six amino acids and two identical chromophoric groups that coordinate copper. (Abstract shortened by UMI.)...