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Electrochemical Evidence That Pyranopterin Redox Chemistry Controls the Catalysis of Yedy, a Mononuclear Mo Enzyme

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Electrochemical Evidence That Pyranopterin Redox Chemistry Controls the Catalysis of Yedy, a Mononuclear Mo Enzyme Electrochemical evidence that pyranopterin redox chemistry controls the catalysis of YedY, a mononuclear Mo enzyme Hope Adamsona, Alexandr N. Simonovb, Michelina Kierzekc, Richard A. Rotheryc, Joel H. Weinerc, Alan M. Bondb, and Alison Parkina,1 aDepartment of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom; bSchool of Chemistry, Monash University, Clayton, VIC 3800, Australia; and cDepartment of Biochemistry, University of Alberta, Edmonton, AB T6G 2H7, Canada Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved October 13, 2015 (received for review August 25, 2015) A long-standing contradiction in the field of mononuclear Mo explores the possibility that ligand-based redox chemistry plays a enzyme research is that small-molecule chemistry on active-site mimic role in YedY catalysis. compounds predicts ligand participation in the electron transfer YedY has been structurally characterized via both X-ray crys- reactions, but biochemical measurements only suggest metal-cen- tallography and X-ray absorption spectroscopy (XAS) (3, 8, 9). In tered catalytic electron transfer. With the simultaneous measurement most mononuclear Mo enzymes, heme groups and iron sulfur of substrate turnover and reversible electron transfer that is provided clusters are found within the same protein as the Mo center, but the by Fourier-transformed alternating-current voltammetry, we show only metal site in YedY is Mo, making this enzyme a helpfully that Escherichia coli YedY is a mononuclear Mo enzyme that recon- simple system for studying redox chemistry (Fig. 1) (1, 3). Within ciles this conflict. In YedY, addition of three protons and three elec- the active site, the X-ray structure was interpreted to show Mo(V) trons to the well-characterized “as-isolated” Mo(V) oxidation state is in a square pyramidal environment (3), identical to other members needed to initiate the catalytic reduction of either dimethyl sulfoxide of the “sulfite oxidase” family of mononuclear Mo enzymes. In or trimethylamine N-oxide. Based on comparison with earlier studies contrast, XAS has suggested a pseudooctahedral Mo center (8, 9) and our UV-vis redox titration data, we assign the reversible one- with an additional O (from Glu104) or N (from Asn45) axial ligand CHEMISTRY proton and one-electron reduction process centered around +174 mV coordinating trans to the apical oxo group (Fig. 1) (8). The equa- vs. standard hydrogen electrode at pH 7 to a Mo(V)-to-Mo(IV) conver- torial ligation is provided by one oxygen-containing ligand and three sion but ascribe the two-proton and two-electron transition occurring sulfur donor atoms, one provided by cysteine (Cys102) and two at negative potential to the organic pyranopterin ligand system. We from the pyranopterin cofactor, whichbindstotheMoinabidentate predict that a dihydro-to-tetrahydro transition is needed to generate fashion via the enedithiolate side chain (3). the catalytically active state of the enzyme. This is a previously A 2012 computational study provided evidence that two dif- unidentified mechanism, suggested by the structural simplicity of ferent oxidation states can be accessed by protein-bound YedY, a protein in which Mo is the only metal site. pyranopterin ligands (10). Conformational analysis and electronic BIOCHEMISTRY structure calculations were used to assign redox states to the Fourier-transformed alternating-current voltammetry | mononuclear pyranopterin ligands in all known mononuclear Mo enzyme molybdenum enzyme | protein film electrochemistry | pyranopterin | YedY structures (10). It was concluded that, although enzymes from the sulfite oxidase family (such as YedY) contain pyranopterin ligands ost living species require a Mo enzyme (1), and apart from in the “dihydro” form, the xanthine dehydrogenase family of enzymes Mnitrogenase, all of these Mo-containing proteins are part of the large family of “mononuclear Mo” enzymes. The general Significance ability of mononuclear Mo enzymes to catalyze two-electron ox- ygen atom transfer reactions has been attributed to the Mo(IV)/ The mononuclear Mo enzymes are ubiquitous throughout life, Mo(V)/Mo(VI) oxidation state cycling of the active site, and this and the notion that their activity arises from Mo(VI/V/IV) redox mechanism is a common part of undergraduate syllabuses (1, 2). Escherichia coli cycling is a central dogma of bioinorganic chemistry. We prove YedY is a mononuclear Mo enzyme (3), and that YedY, a structurally simple mononuclear Mo enzyme, oper- based on sequence homology, the majority of sequenced Gram- – ates via a strikingly different mechanism: the catalytically active negative bacterial genomes encode a YedY-like protein (3 5). state is generated from addition of three electrons and three Uniquely for a mononuclear Mo enzyme, it has not been possible protons to the Mo(V) form of the enzyme, suggesting for the first to form the YedY Mo(VI) state in experiments using ferricyanide time (to our knowledge) that organic-ligand–based electron as an oxidizing agent, and an unusually positive reduction po- + transfer reactions at the pyranopterin play a role in catalysis. tential for the Mo(V/IV) transition [ 132 mV vs. standard hy- We showcase Fourier-transformed alternating-current voltam- drogen electrode (SHE) at pH 7] was determined from EPR metry as a technique with powerful utility in metalloenzyme experiments (6). Although the physiological substrate of YedY is studies, allowing the simultaneous measurement of redox ca- unknown, a possible role in the reduction of reactive nitrogen talysis and the underlying electron transfer reactions. species is suggested by experiments on the pathogen Campylo- bacter jejuni , where deletion of the Cj0379 YedY homolog gen- Author contributions: H.A. and A.P. designed research; H.A. performed research; M.K., R.A.R., erated a mutant that is deficient in chicken colonization and has a J.H.W., and A.M.B. contributed new reagents/analytic tools; H.A. and A.N.S. analyzed data; nitrosative stress phenotype (4). YedY catalysis can be assayed by and H.A., A.N.S., R.A.R., J.H.W., A.M.B., and A.P. wrote the paper. measuring the two-electron reduction of either dimethyl sulfoxide The authors declare no conflict of interest. (DMSO) or trimethylamine N-oxide (TMAO) (3), but the in- This article is a PNAS Direct Submission. accessibility of the YedY Mo(VI) means the enzyme mechanism Data deposition: The graphical data reported in this paper have been deposited with the does not proceed via the common two-electron Mo-redox cycle. University of York Library (www.york.ac.uk/library/info-for/researchers/datasets/). In small-molecule analogs of mononuclear Mo enzymes, the 1To whom correspondence should be addressed. Email: [email protected]. “ ” pterin ligands are described as noninnocent, meaning that the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. redox processes could be ligand or metal based (7). This study 1073/pnas.1516869112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1516869112 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 data to give pure Faradaic data. The baseline-subtracted signals thus obtained are scaled by a 20-fold multiplication factor in Fig. 2A. From Fig. 2A, the integrated area of the baseline-subtracted negative potential process, centered around −250 mV, is ∼1.8 times larger than the integrated area of the baseline-subtracted positive potential process centered around +170 mV. We therefore conclude that al- most twice as many electrons are passed in the negative potential redox transition relative to the positive potential redox transition. As shown in SI Appendix,Fig.S2B, the redox transition measured for YedY at positive potential is well modeled by a Nernstian one- electron process (equivalent to a peak width at half-height, δ,of 90 mV) with a pH 7 midpoint potential Em, 7 =+174 ± 4mV.We attribute this process to the Mo(V/IV) redox transition. Our as- signment of the positive potential redox process as a metal-based Fig. 1. Structure of Escherichia coli YedY. (A and B) The protein structure, PDB ID code 1XDQ (3). (C) The active site in the as-isolated Mo(V) state containing the dihydro form of pyranopterin. contain the two-proton, two-electron more reduced “tetrahydro” form of the pyranopterin (10). Traditionally, redox potential measurements of enzymes have required substrate-free conditions to either permit a solution equilibrium to be established (spectroscopic redox titrations) or to prevent catalytic signals from masking the noncatalytic response (film electrochemistry). Fourier-transformed alternating-current voltammetry (FTacV) is a technique that offers the ability to measure catalytic chemical redox reactions and reversible electron transfer processes in a single experiment (11, 12). In the FTacV measurement, a large-amplitude sine wave of frequency f is superimposed on a linear voltage–time sweep (11, 13–15) and the resulting current–time response is measured and then Fourier transformed (FT) into the frequency domain to give a power spectrum of harmonic contributions at frequencies f,2f,3f, etc. Band selection of the individual harmonics followed by inverse FT resolves the data back into the time domain. The higher harmonic components only arise from fast, reversible redox reactions, de- void of catalysis and baseline contributions, but the aperiodic (dc) component (f = 0) gives the same catalytic information as a tra- ditional dc cyclic voltammetry (dcV) experiment, and can there- fore show catalytic turnover (13, 14, 16). In this study, we both discover previously unidentified mono- nuclear Mo enzyme redox chemistry as well as demonstrate the significant advantages of using FTacV to probe the mechanism of a redox active enzyme. Results Electrochemical Observation of Two Redox Transitions by YedY. We prove that YedY can reversibly form three different oxidation states, i.e., the enzyme can undergo two different redox transitions. This is shown in Fig. 2A, which contains dcV YedY electrochemical data measured under conditions of pH 7 and 25 °C.
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