Spectroscopic and Computational Insight Into the Activation of O2 by the Mononuclear Cu Center in Polysaccharide Monooxygenases
Total Page：16
File Type：pdf, Size：1020Kb
Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases Christian H. Kjaergaarda, Munzarin F. Qayyuma, Shaun D. Wonga, Feng Xub, Glyn R. Hemsworthc, Daniel J. Waltonc, Nigel A. Youngd, Gideon J. Daviesc, Paul H. Waltonc, Katja Salomon Johansene, Keith O. Hodgsona,f, Britt Hedmanf, and Edward I. Solomona,f,1 aDepartment of Chemistry, Stanford University, Stanford, CA 94305; bNovozymes, Inc., Davis, CA 95618; cDepartment of Chemistry, University of York, York YO10 5DD, United Kingdom; dDepartment of Chemistry, University of Hull, Kingston upon Hull HU6 7RX, United Kingdom; eNovozymes A/S, 2880 Bagsværd, Denmark; and fStanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Stanford, CA 94309 Contributed by Edward I. Solomon, May 6, 2014 (sent for review March 18, 2014) Strategies for O2 activation by copper enzymes were recently ex- major chitinases’ degradation of chitin (6, 7, 10–13). Enzymes in panded to include mononuclear Cu sites, with the discovery of the the latest discovered subclass, AA11, are fungal enzymes, where copper-dependent polysaccharide monooxygenases, also classified the currently lone characterized example uses chitin as a substrate as auxiliary-activity enzymes 9–11 (AA9-11). These enzymes are find- (8). Studies by Vaaje-Kolstad et al. (6) and Beeson et al. (14) ing considerable use in industrial biofuel production. Crystal struc- have shown that AA10 and AA9 introduce a single oxygen atom tures of polysaccharide monooxygenases have emerged, but originating from dioxygen into the respective chitin and cellulose experimental studies are yet to determine the solution structure of degradation products. Although little is known about the reaction the Cu site and how this relates to reactivity. From X-ray absorption mechanism of these enzymes, it is believed that this does not near edge structure and extended X-ray absorption fine structure simply involve Fenton-type chemistry producing reactive oxygen spectroscopies, we observed a change from four-coordinate Cu(II) species but rather a more controlled reaction involving a to three-coordinate Cu(I) of the active site in solution, where three Cu-oxygen intermediate (14–16). Cu-loaded crystal structures protein-derived nitrogen ligands coordinate the Cu in both redox have been solved for all three subclasses of AA9-11 enzymes (7, states, and a labile hydroxide ligand is lost upon reduction. The spec- 8, 17–19), highlighting interesting similarities and differences. troscopic data allowed for density functional theory calculations of Common for the three classes is a flat protein surface that har- an enzyme active site model, where the optimized Cu(I) and (II) struc- bors the Cu active site, as illustrated for an AA9 enzyme in Fig. tures were consistent with the experimental data. The O reactivity 2 1, Left. In AA9 PMOs, the flat surface is proposed to interact of the Cu(I) site was probed by EPR and stopped-flow absorption with the long-chained substrates via a number of primarily aro- spectroscopies, and a rapid one-electron reduction of O2 and regen- eration of the resting Cu(II) enzyme were observed. This reactivity matic amino acids (18, 19). In the active Cu site, two histidines was evaluated computationally, and by calibration to Cu-superoxide provide three nitrogen ligands, two from N-His and one from the model complexes, formation of an end-on Cu-AA9-superoxide spe- terminal amine (Fig. 1, Right), in a configuration that has been cies was found to be thermodynamically favored. We discuss how termed a histidine brace (7). An axially oriented tyrosine (Tyr) is ∼ this thermodynamically difficult one-electron reduction of O2 is en- highly conserved in the AA9 and AA11 enzymes 3 Å from the abled by the unique protein structure where two nitrogen ligands Cu, and hydrogen bonded to an oxygen from a glutamine or from His1 dictate formation of a T-shaped Cu(I) site, which provides glutamate side chain (Fig. 1, Right). In the AA10 enzymes, the an open coordination position for strong O2 binding with very little reorganization energy. Significance BIOCHEMISTRY X-ray absorption spectroscopy | DFT | dioxygen activation | biofuels Activation of the O-O bond in dioxygen is difficult but funda- mental in biology. Nature has evolved several strategies to u is an important metal cofactor in a number of enzymes that achieve this, often including copper as an enzyme cofactor. Cactivate dioxygen for reactivity. Several classes with either Copper-dependent enzymes usually use more than one metal binuclear or trinuclear copper active sites have been identified to activate O2 by multielectron reduction, but recently it was and their different strategies for O2 activation have been eluci- discovered that cellulose and chitin degrading polysaccharide CHEMISTRY dated (1). These include the multicopper oxidases that use three monooxygenase enzymes use only a single Cu center for ca- Cu ions to reduce O2 to water with very little overpotential (2, 3), talysis, in a reaction that is of great interest to the biofuel in- the coupled binuclear Cu enzymes that are involved in dioxygen dustries. To understand this reactivity, we have determined the transport and monooxygenase reactivity (4), and the noncoupled solution structures of both the reduced and oxidized Cu site, binuclear Cu monooxygenases that activate O2 for hydroxylation and determined experimentally and computationally how this of peptides and hormones (5). Recently, a class of oxygen acti- site is capable of facile O2 activation by a thermodynamically vating enzymes with a single copper center has been identified, difficult one-electron reduction, via an inner-sphere Cu- the polysaccharride monooxygenases [PMOs; often termed lytic superoxide intermediate. polysaccharide monooxygenases (LPMOs), reflecting their abil- ity to break polysaccharides chains and loosen crystalline struc- Author contributions: C.H.K. and E.I.S. designed research; C.H.K., M.F.Q., and S.D.W. per- – = formed research; C.H.K., M.F.Q., S.D.W., F.X., G.R.H., D.J.W., N.A.Y., G.J.D., P.H.W., K.S.J., ture] (6 8), or AA9 to 11 enzymes (AA auxiliary activity) in K.O.H., B.H., and E.I.S. analyzed data; C.H.K. and E.I.S. wrote the paper; and F.X. expressed the Carbohydrate-Active enZYmes (CAZy) database (9). AA9 and purified protein. enzymes [formerly glycoside hydrolases family 61 (GH61s)] are Conflict of interest statement: F.X. and K.S.J. are employees of Novozymes, which is fungal enzymes that can enhance major cellulases’ enzymatic a commercial supplier of industrial enzymes including (L)PMOs. degradation of cellulose (hence “auxiliary activity”), whereas 1To whom correspondence should be addressed. E-mail: [email protected]. AA10 enzymes [formerly carbohydrate binding module family 33 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (CBM33)] are predominantly bacterial enzymes that can enhance 1073/pnas.1408115111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1408115111 PNAS | June 17, 2014 | vol. 111 | no. 24 | 8797–8802 Downloaded by guest on September 25, 2021 a four-coordinate Cu(II) to a three-coordinate T-shaped Cu(I) site structure. We note that the beat pattern and Fourier trans- form of Cu(II)-AA9 closely resemble that of the oxidized form of copper particulate methane monooxygenase (pMMO) (23, 24), the only other Cu enzyme known to use the histidine brace (6, 19). In contrast to Cu-pMMO, however, the simulation for Cu(II)-AA9 does not require a second Cu ion to be included in the set of nearby scattering atoms. Cryoreduction of Cu(II)-AA9. To probe the temperature dependence of the reduction-induced change in the coordination environ- ment of Cu-AA9, cryoreduction (at 77 K) of Cu(II)-AA9, fol- lowed by XANES and EXAFS evaluation, was performed. A Fig. 1. AA9 from T. aurantiacus (modified from PDB: 3ZUD). Surface view Cu(II)-AA9 sample was prepared in a cryocell, frozen in liquid (Left) and active site (Right) highlighting six selected residues surrounding nitrogen, and subsequently exposed to a γ-emitting 137Cs source the Cu ion (gold sphere). that produces free electrons in the cryosolvent. The extent of cryoreduction of Cu(II)-AA9 was monitored by the disappear- ance of the characteristic Cu(II)-AA9 EPR spectrum (7) axial Tyr is replaced with a highly conserved phenylalanine. In (SI Appendix, Figs. S1A and S2). After 110 h of exposure time addition to these amino acids, water derived molecules are often (∼5.9-Mrad total dose), the enzyme was ∼75% reduced. The refined in the vicinity of the Cu ion, but it is not known whether XANES spectrum of the ∼75% reduced Cu-AA9 (Fig. 4, green these coordinate to the Cu in solution. Although the crystal trace) has a prominent 8984-eV feature that further increases by structures provide valuable information, they do not determine exposure to the synchrotron beam with the final spectrum (black the detailed environment of the Cu ion in solution, which is trace) estimated to be ∼90% reduced, compared with the solu- critical in elucidating the mechanistic properties of the PMOs. tion reduced enzyme (red trace, from Fig. 2). The shape of the This is partly due to the fact that oxidized Cu readily undergoes rising edge in the ∼90% cryoreduced sample is similar to that of photoreduction upon exposure to X-rays, often accompanied by the solution-reduced peak. Further, the EXAFS data and simu- changes in the coordination environment of the metal (17, 20). lation (SI Appendix, Fig. S3 and Table S1) of the cryoreduced Cu- The PMO superfamily is of considerable importance in the AA9 sample are consistent with a mixture of the oxidized and developing area of second generation (recalcitrant polysaccharide) reduced forms, with the reduced form dominant.