Broadus R M, Haddock J D
Department of Microbiology, Southern Illinois University, Carbondale, IL 62901, USA.
Arch Microbiol. 1998 Aug;170(2):106-12. doi: 10.1007/s002030050621.
ferredoxinBPH oxidoreductase (reductaseBPH) of biphenyl 2, 3-dioxygenase was purified over 47-fold to homogeneity with a yield of 41% from cell extract of Pseudomonas sp. strain LB400. ReductaseBPH transfers reducing equivalents from NADH to the catalytic oxygenase component (ISPBPH) via a ferredoxin (ferredoxinBPH) during the oxidation of biphenyl to cis-biphenyl 2,3-dihydrodiol. ReductaseBPH was a monomer with a molecular weight of 43,600 as determined by electrophoresis under denaturing conditions. Gel filtration column chromatography gave a molecular weight of 41,500 for native reductaseBPH. The absorbance spectrum of the protein in its oxidized state had maxima at 271 nm, 376 nm and 448 nm with shoulders at 422 nm and 476 nm. The peak around 448 nm was partially bleached upon reduction with NADH under anoxic conditions. ReductaseBPH contained 0.89 mol FAD/mol protein. ReductaseBPH was required for oxidation of biphenyl to cis-biphenyl 2,3-dihydrodiol by ISPBPH and ferredoxinBPH. Potassium ferricyanide, 2, 6-dichlorophenolindophenol (DCPIP), nitrobluetetrazolium and cytochrome c served as artificial electron acceptors. Reduction of cytochrome c was dependent upon the presence of ferredoxinBPH. The fastest rate of DCPIP reduction occurred at pH 7.2 and 32 degrees C. The apparent Km for NADH and NADPH in the DCPIP assay were 58 microM and 156 microM, respectively. Vmax was 3,120 U mg-1 for NADH and 1, 140 U mg-1 for NADPH. NADH is most likely the physiological electron donor for oxidation of biphenyl and polychlorinated biphenyls.
从假单胞菌属菌株LB400的细胞提取物中纯化出联苯2,3 -双加氧酶的NADH：铁氧化还原蛋白BPH氧化还原酶（还原酶BPH），纯化倍数超过47倍，达到同质，产率为41%。在联苯氧化为顺式联苯2,3 -二氢二醇的过程中，还原酶BPH通过铁氧化还原蛋白（铁氧化还原蛋白BPH）将还原当量从NADH转移至催化加氧酶组分（ISPBPH）。在变性条件下通过电泳测定，还原酶BPH是一种分子量为43,600的单体。凝胶过滤柱色谱法测得天然还原酶BPH的分子量为41,500。该蛋白氧化态的吸收光谱在271nm、376nm和448nm处有最大值，在422nm和476nm处有肩峰。在缺氧条件下用NADH还原后，448nm附近的峰部分褪色。还原酶BPH每摩尔蛋白含有0.89摩尔FAD。ISPBPH和铁氧化还原蛋白BPH将联苯氧化为顺式联苯2,3 -二氢二醇需要还原酶BPH。铁氰化钾、2,6 -二氯酚靛酚（DCPIP）、硝基蓝四唑和细胞色素c作为人工电子受体。细胞色素c的还原依赖于铁氧化还原蛋白BPH的存在。DCPIP还原的最快速率出现在pH 7.2和32℃时。在DCPIP测定中，NADH和NADPH的表观Km分别为58μM和156μM。NADH很可能是联苯和多氯联苯氧化的生理电子供体。
