Dioxygen reduction, with incorporation of one atom into organic substrate, the second into water, designated a monoxygenase reaction, is catalyzed by a variety of substrate-reactive heme-and flavo-proteins. Our work in the current year has focused on the former, a specific P450 cytochrome, P450cam (cytochrome m). Special effort devoted to structural modification of the cyt m and iron sulfide reductant-effector (putidaredoxin, Pd) has enhanced our understanding of their role and specific steps of the methylene hydroxylation. Studies emerging within the year from other laboratories with the adrenal and hepatic P450 hydroxylases have confirmed and extended earlier predictions of the generalized validity of the microbial model we have provided as pure proteins in quantity. We have determined the primary structure of Pd and the role of the single tryptophan, C-terminal, and the penultimate Gln in the cyt m-Pd complex. The KD is 3 micron M as contrasted to 150 micron M for the des Trp-Gln. Catalysis of both steady state and cyt m02rs product release exhibit the same activity ratio. The native cytochrome in crystalline form or in solution undergoes the reaction sequence for product formation in the presence of chemical reductant-effectors. Cyt m modified by the derivatiziation of 4 of the 6 cysteine residues with N-ethyl-maleimide, NEM, leads to the derivatization of 4 of the 6 cysteine residues with N-ethyl-maleimide, NEM, leads to the derivative cyt m NES4, which retains the product- forming capacity and EPR signal of the native protein but lacks the spectral and redox potential shifts on substrate addition. It also shows a decreased substrate affinity from 1 micron M to 100 micron M. The role of superoxide and singlet oxygen in the spontaneous decay of cyt m02rs has been shown. Expertise developed in the heme protein studies for rapid reaction kinetics at low temperature and use of resonant spectral probes will be extended from cyt m to Mb02 and MbCO. The role of copper and iron in dioxygen reductases and oxygenation initiated in the current year will be continued to further detail molecular understanding.
NIH ExPorter
NIH ExPorter