PubMed · 5656646
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1968-06-29. Evergreen enzyme.. https://doi.org/10.1038/2181202a0
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Model building studies have intimated a role for aspartic acid 301 in the substrate binding of cytochrome P450 2D6 (CYP2D6). We have tested this hypothesis by generating a range of CYP2D6 mutants substituting a variety of amino acids at this site. The mutant proteins, which included substitution with a negatively charged glutamic acid residue or neutral asparagine, alanine, or glycine residues, were expressed in Saccharomyces cerevisiae. In addition, a mutant where aspartic acid 301 was deleted was also tested. All the mutants expressed approximately equivalent amounts of recombinant apoprotein and, apart from the alanine 301 and the aspartic acid 301 deletion mutants, gave carbon monoxide difference spectra of similar magnitude to the wild type. In the cases of the alanine and deletion mutants, the amount of holoprotein was significantly reduced or absent relative to the amount of apoprotein, indicating restricted heme incorporation. The glutamic acid mutant was shown to have similar catalytic properties to the wild type enzyme toward the substrates debrisoquine and metoprolol; however, some differences in regioselectivity and ligand binding were observed. The mutants containing neutral amino acids at position 301 exhibited marked reductions in catalytic activity. At low substrate concentrations little, if any, activity toward debrisoquine and metoprolol was measured. However, at a higher substrate concentration (2 mM) some activity was observed (about 10-20% of wild type levels). Consistent with the above findings, the debrisoquine-induced spin changes in the mutant proteins were markedly reduced. These data collectively demonstrate that aspartic acid 301 plays an important role in determining the substrate specificity and activity of CYP2D6 and provide experimental evidence supporting the role of this amino acid in forming an electrostatic interaction between the basic nitrogen atom in CYP2D6 substrates and the carboxylate group of aspartic acid 301.
A Photosystem I (PS I) complex reconstituted with PsaC-C51D (aspartate in lieu of cysteine in position 51) shows light-induced EPR signals with g values, line widths, and photoreduction behavior characteristic of FB. Contrary to an earlier report, a [3Fe-4S] cluster was not located in the reconstituted PS I complex. Instead, a second set of resonances with g values of 2.044, 1.942, and 1.853 becomes EPR-visible when the C51D-PS I complex is measured at 4.2 K. This fast relaxing center, termed FA' is likely to represent a [4Fe-4S] cluster in the mixed ligand (3Cys.1Asp) site. Redox studies show that the Em of FA' and FB are -630 mV and -575 mV, respectively. Room temperature optical studies support the presence of two functioning electron acceptors subsequent to Fx, and NADP+ photoreduction rates mediated by ferredoxin and flavodoxin are nearly equivalent to the wild type. In addition to [3Fe-4S] clusters and S = 1/2 ground state [4Fe-4S] clusters, the free PsaC-C51D protein shows resonances near g = 5.5, which may represent a population of high spin (S = 3/2) [4Fe-4S] clusters in the mixed ligand FA' site. Similar to the C14D-PS I mutant complex, it is proposed that the P700-Fx core selectively rebinds those free PsaC-C51D proteins that contain two [4Fe-4S] clusters. These studies show that primary photochemistry and electron transfer rates in PS I are relatively unaffected by the presence of a highly reducing, mixed ligand cluster in the FA' site.
We have measured proton release into the medium after proton transfer from the retinal Schiff base to Asp85 in the photocycle and the C = O stretch bands of carboxylic acids in wild type bacteriorhodopsin and the E204Q and E204D mutants. In E204Q, but not in E204D, the normal proton release is absent. Consistent with this, a negative band in the Fourier transform infrared difference spectra at 1700 cm-1 in the wild type, which we now attribute to depletion of the protonated E204, is also absent in E204Q. In E204D, this band is shifted to 1714 cm-1, as expected from the higher frequency for a protonated aspartic than for a glutamic acid. Consistent with their origin from protonated carboxyls, the depletion bands in the wild type and E204D shift in D2O to 1690 and 1703 cm-1, respectively. In the protein structure, Glu204 seems to be connected to the Schiff base region by a chain of hydrogen-bonded water. As with other residues closer to the Schiff base, replacement of Glu204 with glutamine changes the O-H stretch frequency of the bound water molecule near Asp85 that undergoes hydrogen-bonding change in the photocycle. The results therefore identify Glu204 as XH, the earlier postulated residue that is the source of the released proton during the transport, and suggest that its deprotonation is triggered by the protonation of Asp85 through a network that contains water dipoles.