The crystal structure of a ternary protein complex has been determined at 2.4 angstrom resolution. The complex is composed of three electron transfer proteins from Paracoccus denitrificans , the quinoprotein methylamine dehydrogenase, the blue copper protein amicyanin, and the cytochrome c 551i . The central region of the c 551i is folded similarly to several small bacterial c-type cytochromes; there is a 45-residue extension at the amino terminus and a 25-residue extension at the carboxyl terminus. The methylamine dehydrogenase-amicyanin interface is largely hydrophobic, whereas the amicyanin-cytochrome interface is more polar, with several charged groups present on each surface. Analysis of the simplest electron transfer pathways between the redox partners points out the importance of other factors such as energetics in determining the electron transfer rates.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCrystal structure of an electron-transfer complex between methylamine dehydrogenase and amicyaninLongyin Chen, Rosemary Durley, Barbara J. Poliks, Kensaku Hamada, Zhiwei Chen, F. Scott Mathews, Victor L. Davidson, Yoshinori Satow, Eric Huizinga, and Cite this: Biochemistry 1992, 31, 21, 4959–4964Publication Date (Print):June 1, 1992Publication History Published online1 May 2002Published inissue 1 June 1992https://doi.org/10.1021/bi00136a006RIGHTS & PERMISSIONSArticle Views217Altmetric-Citations125LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts
Flavocytochrome b2 from Saccharomyces cerevisiae couples l-lactate dehydrogenation to cytochrome c reduction. The crystal structure of the native yeast enzyme has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837−863] as well as that of the sulfite adduct of the recombinant enzyme produced in Escherichia coli [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303−313]; several key active site residues were identified. In the sulfite adduct crystal structure, Arg289 adopts two alternative conformations. In one of them, its side chain is stacked against that of Arg376, which interacts with the substrate; in the second orientation, the R289 side chain points toward the active site. This residue has now been mutated to lysine and the mutant enzyme, R289K-b2, characterized kinetically. Under steady-state conditions, kinetic parameters (including the deuterium kinetic isotope effect) indicate the mutation affects kcat by a factor of about 10 and kcat/KM by up to nearly 102. Pre-steady-state kinetic analysis of flavin and heme reduction by lactate demonstrates that the latter is entirely limited by flavin reduction. Inhibition studies on R289K-b2 with a range of compounds show a general rise in Ki values relative to that of wild-type enzyme, in line with the elevation of the KM for l-lactate in R289K-b2; they also show a change in the pattern of inhibition by pyruvate and oxalate, as well as a loss of the inhibition by excess substrate. Altogether, the kinetic studies indicate that the mutation has altered the first step of the catalytic cycle, namely, flavin reduction; they suggest that R289 plays a role both in Michaelis complex and transition-state stabilization, as well as in ligand binding to the active site when the flavin is in the semiquinone state. In addition, it appears that the mutation has not affected electron transfer from fully reduced flavin to heme, but may have slowed the second intramolecular ET step, namely, transfer from flavin semiquinone to heme b2. Finally, the X-ray crystal structure of R289K-b2, with sulfite bound at the active site, has been determined to 2.75 Å resolution. The lysine side chain at position 289 is well-defined and in an orientation that corresponds approximately to one of the alternative conformations observed in the structure of the recombinant enzyme−sulfite complex [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303−313]. Comparisons between the R289K-b2 and wild-type structures allow the kinetic results to be interpreted in a structural context.
Crystals of [Pt(Et 4 dien)I]I, where Et 4 dien = (C 2 H 5 ) 2 NC 2 H 4 NHC 2 H 4 N(C 2 H 5 ) 2 , have been prepared. A three dimensional crystal structure analysis has been carried out in a monoclinic unit cell having a = 12.565(8), b = 7.576(6), c = 21.040(15) Å, β = 91.12(2)° and space group P2 1 /c, Z = 4. Full-matrix least-squares using 1982 independent, observed reflections converged at R = 0.067. The platinum atom has square-planar co-ordination. The orientation of the four ethyl groups with respect to the plane of the ligand is similar to that observed in related molecules, in spite of a lack of similarity of the molecular packing. The iodide ion appears to be hydrogen bonded to the ligand.
The structure of a ternary complex between methylamine dehydrogenase (MADH), amicyanin and cytochrome c551i, all from Paracoccus denitrificans has been determined at 2.4 Å resolution. MADH and amicyanin associate so that the exposed edges of Tip 108 of TTQ and the His 95 ligand of copper are juxtaposed. Amicyanin and cytochrome c551i associate so that one edge of the β-sandwich of amicyanin is in contact with a chain segment of the cytochrome close to the heme propionates. The distance from the catalytically active quinone oxygen of TTQ to the copper is 16.8 Å and from the copper to the iron is 24.8 Å, respectively. Two efficient paths for electron flow from TTQ to copper were found, one passing through Tip 108 of MADH. Two paths from copper to iron were also found, one through the cysteine and one through the methionine ligand to copper, which converge at Tyr 30 of amicyanin.
The structure of bovine liver cytochrome b(5), a soluble 93-residue proteolytic fragment of a 16 kDa membrane-bound hemoprotein, initially solved at 2.0 A resolution, has been refined at 1.5 A using data collected on a diffractometer. Refinement to 2.0 A resolution used the Hendrickson-Konnert procedure PROLSQ and was then extended to 1.5 A resolution using the program PROFFT. Only residues 3-87 could be identified in the model and these residues together with 93 water molecules gave an agreement factor of R = 0.161 for data in the resolution range 1.5-5 A. The structure was finally refined using the program X-PLOR, which enabled alternate conformers to be modelled for several surface side chains. Residues 1 and 2 at the amino terminus of the protein and residue 88 near the carboxyl terminus could be identified from these electron-density maps. However the remaining disordered carboxy-terminal residues could not successfully be included in the model. A total of 117 solvent molecules were included in the final refinement to give R = 0.164 for the data between 1.5 and 10 A.
Abstract The crystal structure of amicyanin, a cupredoxin isolated from Paracoccus denitrificans , has been determined by molecular replacement. The structure has been refined at 2.0 Å resolution using energy‐restrained least‐squares procedures to a crystallographic residual of 15.7%. The copper‐free protein, apoamicyanin, has also been refined to 1.8 Å resolution with residual 15.5%. The protein is found to have a β ‐sandwich topology with nine β ‐strands forming two mixed β ‐sheets. The secondary structure is very similar to that observed in the other classes of cupredoxins, such as plastocyanin and azurin. Amicyanin has approximately 20 residues at the N‐terminus that have no equivalents in the other proteins; a portion of these residues forms the first β ‐strand of the structure. The copper atom is located in a pocket between the β ‐sheets and is found to have four coordinating ligands: two histidine nitrogens, one cysteine sulfur, and, at a longer distance, one methionine sulfur. The geometry of the copper coordination is very similar to that in the plant plastocyanins. Three of the four copper ligands are located in the loop between β ‐strands eight and nine. This loop is shorter than that in the other cupredoxins, having only two residues each between the cysteine and histidine and the histidine and methionine ligands. The amicyanin and apoamicyanin structures are very similar; in particular, there is little difference in the positions of the coordinating ligands with or without copper. One of the copper ligands, a histidine, lies close to the protein surface and is surrounded on that surface by seven hydrophobic residues. This hydrophobic patch is thought to be important as an electron transfer site.
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