Intramolecular Electron Transfer in the Bacterial Two-Domain

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Intramolecular Electron Transfer in the Bacterial Two-Domain Multicopper Oxidase mgLAC Scot Wherland, Kentaro Miyazaki, and Israel Pecht Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00158 • Publication Date (Web): 29 Apr 2016 Downloaded from http://pubs.acs.org on May 10, 2016

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Biochemistry

Intramolecular Electron Transfer in the Bacterial Two-Domain Multicopper Oxidase mgLAC Scot Wherland#, Kentaro Miyazaki% and Israel Pecht&* #

Department of Chemistry, Washington State University, Pullman, Washington 99164-4630, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 10101 Higashi, Tsukuba Ibaraki 205-8566, Japan & Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel %

AUTHOR INFORMATION Corresponding author *E-mail: [email protected] Phone: (972)-8-934-4020. Notes The authors declare no competing financial interest. Abbreviations: ET, electron transfer; MCO, multicopper oxidase; T1, type 1 copper site; T2, type 2 copper site; T3, type 3 copper site; TNC, trinuclear center, a combination of T2 and the two T3 Cu sites; NHE, normal hydrogen electrode; SLAC, small laccase from Streptomyces coelicolor; AO, ascorbate oxidase; 2-d, 2 domain; 3-d, three domain; EPR, electron paramagnetic resonance; UV/VIS, ultraviolet-visible wavelength spectroscopy; NTA, nitrilotriacetate; Cys, cysteine; His, histidine

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ABSTRACT The kinetics of the intramolecular electron transfer process in mgLAC, a bacterial two-domain MultiCopper Oxidase (MCO) were investigated by pulse radiolysis. The reaction is initiated by CO2− radicals produced in anaerobic, aqueous solutions of the enzyme by microsecond pulses of radiation. A sequence of pulses of CO2− radicals enables examination of reductive half-cycle of the MCO catalysis. This is done by titrations of the Type 1 (T1) Cu(II) site and monitoring the time course and amplitude of its reoxidation by internal electron transfer (ET) to the Type 3 site. Comparison of the internal ET kinetics observed for mgLAC with those of other MCOs that have been studied, by pulse radiolysis, shows that they exhibit rather distinct reactivities. One main cause for the different reactivities is the broad range of T1 copper redox potentials, from the moderate potential of bacterial enzymes to the high potential of fungal laccases, and possibly also reflect evolutionary quaternary structural adaptation of the MCO family to the rather wide range of reducing substrates that they oxidize while maintaining efficient reduction of the common substrate, molecular oxygen.


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Biochemistry

The family of multicopper oxidases (MCOs) is rather widespread in nature. All MCOs share a fundamental set of three distinct copper binding sites: Single electrons are taken up from an exceptionally wide variety of reducing substrates by the “blue” or “Type 1” (T1) copper site and, then delivered intramolecularly to a binding site constituted of three copper ions, designated the trinuclear center (TNC), where dioxygen is reduced to water without release of any intermediate. The TNC is composed of a binding site of two, antiferromagnetically coupled Cu(II) ions designated “Type 3” (T3) and an additional copper ion site designated “Type 2” (T2).1-3 Broadly, there is one distinction between MCOs based on their T1 site reduction potential, the higher potential category (ca. 800 mV vs. NHE) includes the fungal laccases, such as the Trametes hirsuta laccase discussed below. Enzymes in the other MCO category have a T1 potential near +400 mV.4,5 This variation is presumed to be an adaptation to the different reducing substrates utilized. The MCOs are also structurally distinguished by the way they are assembled from two to as many as six cupredoxin domains. This variation probably reflects conditions under which they have evolved.6-8 These interesting structural and reactivity differences, amplified by MCOs’ efficient catalysis of dioxygen reduction to water, has produced considerable interest, both fundamental and practical, in these enzymes, leading to many studies aiming at a detailed understanding of their reaction mechanism(s). Pulse radiolysis enables monitoring directly the internal electron transfer (ET) rates and distributions in several different MCOs.9-13 Investigation of the internal T1 to T3 ET reaction, i.e. the reductive half-cycle of the enzyme’s catalysis has inherent implications for understanding the latter mechanism and may also provide insights into the evolution of the interesting differences among MCOs. For example, the Small Laccase (SLAC) from the bacterium Streptomyces coelicolor, is a 2 cupredoxin domain MCO (2-d MCO)6,7,14 with a trimeric quaternary structure. This 2-d MCO group contains three subgroups (A, B and C) differing in the location of the copper sites in their structure with SLAC belonging to the B subgroup. Studies of the internal electron transfer kinetics and distribution in this enzyme resolved a rather surprising behavior: a cooperative site-site interaction expressed in a tenfold enhancement of the T1 Cu(I) to T3 Cu(II) ET rate as the extent of the enzyme reduction increased. This behavior has not been encountered so far in any other MCO. In view of these interesting structural and redox potentials differences among MCOs, we have now studied the internal ET rates within another trimeric, 2-d MCO, mgLAC, originally identified in a bacterial metagenomic library. The 3-dimensional structure and functional features of oxidized mgLAC have recently been reported establishing its belonging to the 2-d MCO C-subgroup.15 We have investigated the kinetics of reduction of mgLAC employing the pulse-radiolysis method monitoring the electron uptake by the T1 Cu(II) site and its following transfer to the T3 site yielding an equilibrium between the sites. The current results are compared with those of studies of MCOs with different structural properties and redox potentials, from SLAC10 to zucchini squash ascorbate oxidase (AO)9, and the laccase from the fungus Trametes hirsuta.11

EXPERIMENTAL PROCEDURES The gene for mgLAC was found by in silico screening of a metagenomic sequence database for proteins containing a copper-binding motif. The gene was sequenced, introduced into and expressed by Escherichia coli. The expressed protein was purified from cell lysates using a Qiagen (Hilden Germany) Ni-NTA column, as previously described.16 Enzyme properties, including activity with several substrates as measured by oxygen reduction, and both EPR and UV/VIS spectra, were included in Supplementary material (Table S1) of Ref. 15. The ratio of the absorbance at 605 nm to that at 330 nm of the mgLAC samples studied was in the range 0.6-0.7. The pulse radiolysis experiments were carried out using the facility of The Hebrew University in Jerusalem, consisting of a Varian V-7715 linear accelerator and a monitoring system described earlier;12 5 MeV electrons were used with pulse lengths in the range from 0.1 to 1.5 µs. The kinetic measurements of the internal T1-T3 ET reactions were studied under anaerobic conditions in enzyme solutions that contained 10mM sodium dihydrogen phosphate, 100mM sodium formate, the pH was adjusted to values from 5.8 to 9.1 and they were saturated with N2O. Reaction solutions were kept in a rubber-sealed, 10 mm Spectrosil cuvette subjected to three light passes of the probing light. The


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temperature was maintained in the range 5 to 46°C, as monitored by a thermocouple attached to the optical cuvette holder. In aqueous solutions subjected to high-energy electrons, the dominant primary products of interaction with the solvent, water are hydrated electrons and OH radicals.17 These rather reactive species are converted in the above solutions to a milder, more selective agent CO2− radical (reduction potential of -1.80 V vs. SHE)18, by reaction with the solutes, formate ions and N2O as follows: e−aq + N2O + H2O → N2 + OH− + OH OH + HCOO− H2O + CO2−

(1) (2)

Enzyme concentrations were in the 14-82 µM range. All reactions were performed under pseudo-first order conditions, with at least a 10-fold or more excess of oxidized enzyme over reducing radicals. The change in concentration of T1Cu(II) was monitored at 605 nm (Δε605 = 4,890 M−1 cm−1, estimated from the relative absorbance at 605 and 280 nm, the latter extinction coefficient, calculated from the protein’s sequence16, at 280 nm of 59,360 M-1 cm-1 and the assumption that the T1 site is fully occupied, while for T3Cu(II) we estimate Δε330= 2,720 ± 10% M−1 cm−1 from the comparison of reduced and oxidized spectra.

RESULTS Electron uptake and internal transfer-- Under anaerobic conditions, attained after several pulses that consumed adventitious oxygen in N2O saturated, 0.10 M sodium formate and 10 mM phosphate buffer containing solutions, the electron donor CO2− radical was found to react with mgLAC, reducing only the T1Cu(II), with a rate that depends linearly on protein concentration, i.e. in a second-order process with a rate constant of ~109 M-1s-1, Fig. 1. T1Cu(II) + CO2− → T1Cu(I) + CO2

(3)

Following this bimolecular initial step, two well-resolved phases of absorption increase were observed at 605 nm (Fig. 2A and Figs. S1A and B of the Supporting Information) reflecting reoxidation of the T1Cu(I). Since the ET rates of both reoxidation phases were found to be independent of the enzyme concentration or its extent of reduction (see below), they are interpreted as being intramolecular electron equilibration between the T1 and T3 sites: T1Cu(I)T3Cu(II)T3Cu(II) ⇄ T1Cu(II)T3Cu(I)T3Cu(II)

(4)

This assignment is further corroborated by the observed concomitant time-resolved absorption decrease at 330 nm reflecting reduction of T3Cu(II) (Fig. 2B and Figs. S1A and B (Supporting Information). Rate constants of the T1 to T3 ET are independent of the enzyme’s reduction state--We have previously observed that the trimeric, 2 domain MCO SLAC, exhibits a marked dependence of the T1 to T3 ET rate constant on the number of reduction equivalents taken up by the enzyme.10 Hence we now examined the intramolecular (T1⇄T3) ET rate constants of mgLAC and found that within experimental error these do not change as the degree of the enzyme’s reduction increases. As Fig. S2A and B (Supporting Information) illustrate rate constants of the two intramolecular ET phases monitored at both 605 and 330nm were essentially constant up to 90% reduction of the enzyme. The ratio of the total amplitude of the internal ET (the sum of the two oxidation phases at 605 nm (T1) and the two reduction phases at 330 nm (T3) is about 1.5 which compares well with Δε605 / Δε330 ≈ 1.8 derived from the estimates of the change in the extinction coefficients. This result indicates that the absorbance at 330 nm attributed to the spin-coupled T3 Cu(II) ion pair is completely bleached already upon reduction by a single electron. This is further substantiated by both its magnitude and by having it observed before even an average of one reducing equivalent has been taken up by the enzyme.


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As illustrated by Fig. 2A, the 605 nm absorbance does not return to its initial absorption level at the end of the slow reoxidation phase i.e. the T1Cu(I) site is not fully reoxidized. This reflects the establishment of equilibrium between the T3 and T1 sites (cf. reaction 4 above). From the respective absorption amplitudes (initial, direct T1 reduction and the total reoxidation) we calculated an overall apparent equilibrium constant KTOT = 4 ± 1 at 286 K. At other temperatures rather similar equilibrium constants for this process are calculated (4) KTOT = 4 ± 1 (298 K and 319 K). Assuming the two observed phases of T1 Cu(I) reoxidation represent reactions of two populations of molecules, then there are two equilibrium constants, one for each. However, since we cannot evaluate separately the amplitudes of the two T1 reduction phases, one cannot calculate the two independently. The apparent KTOT value decreases to about 1 at the end of the titration, but the value of 4 best represents the oxidized protein and is used below. Temperature dependence of the intramolecular ET rate constants--Rate constants of the two intramolecular ET processes were determined at five different temperatures (pH 7.5) all in N2O saturated, anaerobic conditions. Results monitored at 605nm, are presented in an Eyring plot below in Fig. 3 (k298 = 110 ± 4 s-1 and 3.0 ± 0.5 s-1, respectively at pH 7.5). The activation parameters, calculated by fitting data to the Eyring equation, Eq. (5), are ΔH‡ = 42.8 ± 2.3 and 36.5 ± 5.8 kJ mol-1 and ΔS‡ + Rln(κ) = -62 ± 5 and -117 ± 18 J K-1 mol-1 for the fast and slow phases, respectively. In Eq. (5) kB is the Boltzmann constant, T is the absolute temperature, and h is Planck’s constant. The transmission coefficient (κ) is related to β, the distance dependence of electron transfer probability in the Marcus theory, and thus is part of the apparent activation entropy. This is discussed further below.

=

κ exp −

∆ ‡

∆ ‡

+

(5)

Examination of the activation parameters makes it evident that the difference between rates of the two phases is dominated by the less favorable entropy of activation of the slower process, which overcomes the somewhat more favorable enthalpy of activation. pH dependence of intramolecular ET rates--In order to examine whether the two phases of intramolecular ET are due to the presence of two populations with distinct protonation states of the enzyme, pulse radiolysis experiments were carried out at 3 different pH values, 5.6, 7.5, and 9.1. Results at 298 K are shown in Table 1 below. The amplitude ratio between the two intramolecular electron equilibration steps, presented in Table 1 shows that the distribution between the two populations is only moderately affected by pH changes. This apparently excludes protonation/deprotonation of the protein as the main cause for the observed two phases of the T1⇄T3 ET reaction. However, the fast reoxidation phase seems to be most prominent around pH 7.5 while the slow phase dominates at lower and higher pH. The observed limited pH dependence of the rate constants, with maximum around pH 7.5, is in accordance with the reported pH maximum for the enzyme activity at around pH 8 (K. Miyazaki, previously unpublished results). Behavior of the partly cycled enzyme- In order to try resolving potential differences in the internal ET reactions between resting (i.e. “as isolated”) and cycled (pulsed) mgLAC that has been reduced by the CO2− radicals and reoxidized by oxygen, we have used sequential pulses to carry out reductive titrations of the enzyme in the presence of limited but varied O2 concentrations. In the results depicted in Fig. S2A and S2B (Supporting Information), about 17 pulses were required to eliminate the added O2, as compared for example to only 5 in the experiment shown in Supp. Fig. S1A and S1B (Supporting Information). The reaction pattern is the same in both of these cases, namely after the initial fast bimolecular reduction of T1Cu(II) by CO2− the fast and slow T1⇄T3 electron equilibrations are observed with similar amplitudes and rates constants. In another experiment the enzyme was first partially reduced by CO2− radicals, then O2 was added to completely reoxidize it, followed by bubbling with N2O and subjecting it to further reductive pulses. Again, after consuming the small residues of added O2, the same two-reoxidation rate constants with similar amplitudes were observed. Thus, no influence was observed of having oxygen initially present in the solution on the internal ET rate constants measured after the oxygen has been consumed and no evidence was found that the mgLAC is affected by undergoing a reduction/reoxidation cycle. As Fig. 5 ACS Paragon Plus Environment

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S2A (Supporting Information) shows, no further reduction of T1 site or its reoxidation are observed after ca. 3 reducing equivalents have been taken up by the enzyme, as monitored by changes in absorbance at 605 nm. Also noteworthy is that these reaction amplitudes diminish as the titration proceeds, illustrating that upon approaching full reduction of the enzyme molecules, fewer of the CO2− radicals actually reduce the protein. In the absence of electron uptake by the mgLAC T1site, competing side reactions, mainly dimerization to produce oxalate, dissipate these radicals.

DISCUSSION The rather widespread presence of MCOs in nature and the marked increasing interest in them cause new members of this family to be continuously discovered and studied. Though all share very similar copper binding sites and covalent connections between them, the enzymes belong to clearly distinct structural subgroups. Thus, while both SLAC and mgLAC are homotrimeric, 2-d MCOs14, 15 they belong to different subgroups6,7 (Table 2), differing in the location of the T1 Cu. In SLAC the T1 Cu is in the second domain while in mgLAC it is in the first domain. Consequently the T1 Cu ions of the SLAC trimer are localized near the surface of the central cavity while those of mgLAC are positioned on the outside surface. The TNC of both subgroups is located at the monomer-monomer interface and each T3 Cu is coordinated asymmetrically to 2 His ligands from the same chain as the T1 and one from another chain. ET pathways between T1 and T3 Cu ions start from the Cys ligand of the T1 Cu and then go via the His residue preceding the Cys in the sequence to one T3 Cu and the His residue following the Cys to the other T3 Cu (Fig. 4).19 SLAC differs from other MCOs, including mgLAC, in having a tyrosine residue near the TNC that was shown to be oxidized during turnover.20 An additional issue is that crystallographic studies often show partial occupancy of the Cu sites, especially at the T2 site, possibly exacerbated by radiation exposure during the process. Although these variations in occupancy also occur in the mgLAC structures,3 careful consideration of the patterns of occupancy and the reactivity tend to exclude any correlation. The marked differences resolved among MCOs in their internal ET reactivities, an inherent, key step of their catalytic cycles, are of considerable interest.8, 13 Unfortunately knowledge of the three-dimensional structures of the copper binding sites is practically limited to crystals of their apparently oxidized states, and structures produced when these crystals are reduced by the intense synchrotron x-rays, notably under aerobic conditions. These experiments have produced information about a variety of intermediate structures with O and O2 at different positions in the TNC. However, the experimental procedures employed do not establish whether the observed structures are part of the catalytic cycle. The two phases of intramolecular T1 to T3 ET observed here are noteworthy. This reaction pattern has been observed also for other MCOs, notably AO, a dimer of 3-d chains (Table 2), and probably reflects the existence of two populations of the enzyme that might be in equilibrium, yet a slow one, on the time scale of the present experiments. The two reaction phases observed cannot be sequential processes in the same molecule since each pulse yields much less than 1 reduction equivalent per MCO monomer within microseconds, and thus any particular molecule will undergo only a single subsequent T1⇄T3 equilibration process. Furthermore, since no marked difference was observed in the specific rates or the relative amplitudes of the distinct phases as a function of the extent of mgLAC reduction, the two enzyme populations do not reflect distinct reduction states, as observed for the structurally related SLAC.10 A third possibility, that there are different enzyme forms that occur during turnover and as isolated, also seems to be ruled out by the similar rates and amplitudes observed for the resting and cycled enzyme. The two orders of magnitude difference between the two rates of intramolecular ET probably reflects differences at the T3 site. The apparent reorganization energy for the ET processes (λ), of both the T1 and T3 sites, can be calculated using the following equations .20

= 1(10 )exp (−)exp (− "

∆! ∗ = # $1 +

∆ % "

&

∆ ∗

)

'

(6)

(7) 6

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where D is the separation distance between T1 and T3 Cu ions (taken as 1.26 nm, the average of the distances (1.25 and 1.27 nm) in the mgLAC three-dimensional structure (PDB: 4E9V)3, β the distance decay factor (here 10 nm−1)21, ∆G* the activation energy, and ∆G0 the standard free energy change of the ET reaction. The free energy change is estimated from the equilibrium constant determined for the intramolecular ET, KTOT = 4. Employing the two ET rate constants, 110 s-1 and 3 s-1 this calculation gives λ = 132 and 168 kJ (1.4 and 1.7 eV) respectively, similar to values calculated for other MCOs11 but smaller than the value for quinol/amicyanin ET and larger than the value for amicyanin/cytochrome ET in the methylamine dehydrogenase-amicyanin-cytochrome c-551i protein ET complex22. This calculation gives the maximum difference in reorganization energy if the two processes differ only in that parameter and if the coupling between T1 and T3 is adequately represented by the exponential dependence on distance, assumed to be the same for the two ET steps. The mgLAC three-dimensional structure does not provide any option that would allow the two different rate constants, to be correlated with ET paths of the different distance that would be required. However, the difference cannot be due only to different reorganization energies at the T3 sites, since the reorganization energy should be reflected primarily in the activation enthalpy, ΔH‡, and its value for the slower process is actually somewhat lower than that of the faster one. Hence, the difference in rates of the two phases is determined primarily by their activation entropies, with a less favorable value for the slower process of 55 J K−1 mol−1. This might indicate the existence of two distinct electronic couplings between the T1 and T3 sites. An entropic effect on the coupling may originate in differences in structure and or solvation, especially as there is some entropy/enthalpy compensation. Still it seems difficult to generate enough of a change in solvation, affecting the reorganization energy and the driving force, to yield such a difference in reactivity. In contrast, changes in electronic coupling can occur from subtle differences in the structure as elaborated by Solomon et al regarding the T1 to T3 potential pathways.24 The two limiting rate constants observed for the related SLAC also show the same pattern, the slower process has a similar enthalpy but a less favorable entropy of activation. Intramolecular ET in ascorbate oxidase, a three-domain MCO dimer with all Cu sites within each monomer unit, also exhibits two internal ET phases of comparable amplitude. For the fastest phase a rate constant of 200 s-1 was determined at 25 oC, pH 5.5 under anaerobic conditions along with a rate constant of 2 s-1.9 The activation parameters were also similar to those of mgLAC. The fungal laccase from Trametes hirsuta, a 3-d monomer, exhibits a single phase for the T1⇄T3 equilibration process with a moderate 25 s-1 rate constant and activation parameters between the two sets of values from mgLAC (Table 2), in spite of its T1 site having ca. 400 mV higher potential than the bacterial enzymes. An important issue is the fact that the internal ET rates and distributions are investigated in the absence of the common, unique oxidizing substrate of MCOs, dioxygen. Clearly in the presence of O2 many elements of these enzymes’ reactivities would be greatly affected, first and foremost the driving forces of the internal electron transfer. Future studies are planned to pursue these issues. In this context of dioxygen reduction it is of interest to consider the enzymatic activities of mgLAC. As the natural reducing substrate of this MCO is not known, activity has been determined using conventional reductants such as methylsyringate, syringaldehyde, and 2,6-dimethoxyphenol. In all cases the Vmax was much lower than the rates of intramolecular ET equilibration [Eq. (2) above]. Typically the Vmax values were in the range from 0.1 – 0.3 s−1 at 298 K and pH 8.0, the optimum for activity. Thus the rate-limiting step in the catalytic cycles are determined by the examined substrates.15 The use of pulse radiolysis for investigation of the internal ET reactions in redox enzymes, notably here MCOs, enables resolution of the elementary steps of their catalysis and the parameters controlling them. In all MCOs investigated so far, in the absence of the oxidizing substrate O2, a rather small ET equilibrium constant between the T1 and T3 sites has been observed (Table 2). In order to maintain ET from T1 to the TNC with the same or similar equilibrium constant, the MCOs with markedly different T1 potentials must also have adjustments of their T3 potentials. This adjustment possibly reflects the evolutionary adaptation of the MCO family to the needs of oxidizing extremely diverse electron donors with a wide range of reduction potentials, while maintaining the capacity to efficiently reduce one and the same substrate, molecular oxygen. The observation of distinct rate constants for the same enzyme also hints at structural variability, most likely in the vicinity of the TNC, influencing the pattern of reactivity. Thus the apparent similarity of active-sites’ 7 ACS Paragon Plus Environment

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structures in the MCO family obscures significant differences in the kinetics and thermodynamics of their reductive catalytic half-cycle processes, and therefore they may also exhibit distinct differences in their oxidation mechanisms by dioxygen. Differences in the thermodynamics of reduction may be illustrated by results of early studies of the internal electron distribution among the Rhus vernicifera laccase sites using a group of distinct reductants. It has resolved remarkable reductant-dependent differences that were assigned to possible non-equilibrium states involving transition of the T3 site from a cooperative 2-electron acceptor to an uncoupled pair of one-electron acceptors .25

ASSOCIATED CONTENT Supporting Information Figures S1 A and B, and S2 A and B depicting rate constants and amplitudes of internal ET. This material is available free of charge via the Internet at http://pubs.acs.org.


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Malkin, R., and Malmstrom, B. G. (1970) State and function of copper in biological systems, Advances in Enzymology and Related Subjects of Biochemistry 33, 177-&. Reiss, R., Ihssen, J., Richter, M., Eichhorn, E., Schilling, B., and Thony-Meyer, L. (2013) Laccase versus laccase-like multi-copper oxidase: a comparative study of similar enzymes with diverse substrate spectra, Plos One 8, e65633. Komori, H., Sugiyama, R., Kataoka, K., Miyazaki, K., Higuchi, Y., and Sakurai, T. (2014) New insights into the catalytic active-site structure of multicopper oxidases, Acta Crystallographica Section D-Biological Crystallography 70, 772-779. Tepper, A., Aartsma, T. J., and Canters, G. W. (2011) Channeling of electrons within SLAC, the small laccase from Streptomyces coelicolor, Faraday Discussions 148, 161-171. Shleev, S., Christenson, A., Serezhenkov, V., Burbaev, D., Yaropolov, A., Gorton, L., and Ruzgas, T. (2005) Electrochemical redox transformations of T1 and T2 copper sites in native Trametes hirsuta laccase at gold electrode, Biochemical Journal 385, 745-754. Nakamura, K., Kawabata, T., Yura, K., and Go, N. (2003) Novel types of two-domain multicopper oxidases: possible missing links in the evolution, Febs Letters 553, 239-244. Lawton, T. J., Sayavedra-Soto, L. A., Arp, D. J., and Rosenzweig, A. C. (2009) Crystal structure of a two-domain multicopper oxidase implications for the evolution of multicopper blue proteins, Journal of Biological Chemistry 284, 10174-10180. Giardina, P., Faraco, V., Pezzella, C., Piscitelli, A., Vanhulle, S., and Sannia, G. (2010) Laccases: a never-ending story, Cellular and Molecular Life Sciences 67, 369-385. Farver, O., and Pecht, I. (1992) Low activation barriers characterize intramolecular electrontransfer in ascorbate oxidase, Proceedings of the National Academy of Sciences of the United States of America 89, 8283-8287. Farver, O., Tepper, A., Wherland, S., Canters, G. W., and Pecht, I. (2009) Site-site interactions enhances intramolecular electron transfer in Streptomyces coelicolor laccase, Journal of the American Chemical Society 131, 18226-27. Farver, O., Wherland, S., Koroleva, O., Loginov, D. S., and Pecht, I. (2011) Intramolecular electron transfer in laccases, Febs Journal 278, 3463-3471. Farver, O., and Pecht, I. (2007) Elucidation of electron-transfer pathways in copper and iron proteins by pulse radiolysis experiments, In Progress in Inorganic Chemistry, Vol 55, pp 178. Wherland, S., Farver, O., and Pecht, I. (2014) Multicopper oxidases: intramolecular electron transfer and O2 reduction, Journal of Biological Inorganic Chemistry 19, 541-554. Skalova, T., Duskova, J., Hasek, J., Stepankova, A., Koval, T., Ostergaard, L. H., and Dohnaklek, J. (2011) Structure of laccase from Streptomyces coelicolor after soaking with potassium hexacyanoferrate and at an improved resolution of 2.3 angstrom, Acta Crystallographica Section F-Structural Biology and Crystallization Communications 67, 2732. Komori, H., Miyazaki, K., and Higuchi, Y. (2009) X-ray structure of a two-domain type laccase: A missing link in the evolution of multi-copper proteins, Febs Letters 583, 11891195. Komori, H., Miyazaki, K., and Higuchi, Y. (2009) Crystallization and preliminary X-ray diffraction analysis of a putative two-domain-type laccase from a metagenome, Acta Crystallographica Section F-Structural Biology and Crystallization Communications 65, 264266. Hart, E. JI., and Anbar, M. (1970) The Hydrated Electron, Wiley, New York. Klapper, M. H., and Faraggi, M. (1979) Applications of pulse-radiolysis to protein chemistry, Quarterly Reviews of Biophysics 12, 465-519. Messerschmidt, A., Ladenstein, R., Huber, R., Bolognesi, M., Avigliano, L., Petruzzelli, R., Rossi, A., and Finazziagro, A. (1992) Refined crystal-structure of ascorbate oxidase at 1.9 Å resolution, Journal of Molecular Biology 224, 179-205. Marcus, R. A. and Sutin, N. (1985) Electron transfers in chemistry and biology Biochimica et Biophysica Acta 811, 265-322. Gray, H. B. and Winkler, J. R (2003). Electron tunneling through proteins Quarterly Review 9 ACS Paragon Plus Environment

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of Biophysics 36, 537-61. 22. 23.

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Davidson, V. L. (2008) Protein control of true, gated, and coupled electron transfer reactions Accounts of Chemical Research 2008 41 (6), 730-738. Gupta, A., Nederlof, I., Sottini, S., Tepper, A. W. J. W., Groenen, E. J. J., Thomassen, E. A. J., and Canters, G. W. (2012) Involvement of Tyr108 in the enzyme mechanism of the small laccase from Streptomyces coelicolor, Journal of the American Chemical Society 134, 1821318216 Hadt, R. G., Gorelsky, S. I., and Solomon, E. I. (2014) Anisotropic covalency contributions to superexchange pathways in type one copper active sites, Journal of the American Chemical Society 136, 15034-15045. Farver, O., Goldberg, M., Wherland, S. And Pecht, I. (1979) Reductant-dependent electron distribution among redox sites of laccase Proceedings of the National Academy of Sciences of the United States of America 75, 5245-5249.


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Biochemistry

Table 1 Internal T1Cu(I) to T3Cu(II) ET rates and amplitudes at different pHs (25 oC; anaerobic conditions).

pH 5.6 7.5 9.1

kreox (fast) s-1 21 ± 2 110 ± 4 102 ± 5

kreox (slow) s-1 1.2 ± 0.1 3.0 ± 0.5 0.8 ± 0.1

Ampreox(fast):Ampreox(slow) 0.65 : 1 1.10 : 1 0.80 : 1

Table 2 Kinetic parameters for T1⇄T3 ET process in MCOs determined by pulse radiolysis

Enzyme mgLAC SLAC Streptomyces coelicolor AO Curcurbita pepo Fungal laccase Trametes hirsuta

k298 K s-1 110 4 3 8 to 0.4 186 to 5

∆ H‡ kJ/mol 42.8 36.5 25.2 26.2

∆S‡ J/mol K -62 -117 -142 -115

201 2.3 25

9.1 6.8 39.7

-170 -215 -87

1.8 0.4

MCO type

Ref

Trimer of 2-d chains C subgroup TNC at interface between chains Trimer of 2-d chains B subgroup TNC at interface between chains Tyr oxidized during turnover k increases with extent of reduction Dimer, 3-d chains TNC within each monomer Monomer, 3-d chains High potential T1 T1 ligand Met replaced by Phe

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Figure captions Figure 1.Time resolved absorption changes monitored at 605 nm following introduction of a 1.0 µs pulse. Protein concentration is 29.7 µM; temperature 25 oC; optical path length 3.00 cm; pH 7.5. The initial, fast absorption decrease begins at an absorbance of 0 as set by the data collection software, and shows that 1.9 µM T1Cu(II) are reduced with this pulse. Time-base is 0.4 ms for the left panel, the following 4.6 ms for the right. The lower panels display the residuals between the observed data and the calculated red fit line. Figure 2A. Time-resolved absorption changes followed at 605 nm after introduction of a 1.0 µs pulse. Protein concentration is 24.0 µM; temperature 298 K; optical path length 3.00 cm; pH 5.6. The initial, fast absorption decrease begins at an absorbance of 0 as set by the software, and shows that 2.3 µM T1Cu(II) are reduced per pulse. Time base is 0.20 s for the left panel, the following 10 s for the right. The lower panels display the residuals between the observed data and the red fit line. Figure 2B. Time-resolved absorption changes monitored at 330 nm following introduction of a 1.0 µs pulse. All conditions as in Fig. 2A. Figure 3 Eyring plot of intramolecular T1 to T3 ET rate constants of the fast (circles) and slow (diamonds) phases at pH 7.5. ∆H‡ = 42.8 ± 2.3 and 36.5 ± 5.8 kJ mol-1 ∆S‡ = -62 ± 5 and -117 ± 18 J K-1 mol-1 for the faster and slower phases, respectively. An Arrhenius plot (ln(k) vs 1/T) is similarly linear and gives activation energies of 45.2 and 39.0 kJ mol-1. Figure 4 Through bond ET pathway from T1 to T3 copper ions. The direct distance between the T1 and T3 copper ions is 1.25 and 1.27 nm. The entirely covalent bond pathways (green atoms) from the T1 to each T3 copper involve 11 covalent bonds. They both follow the cysteine sulfur T1 Cu ligand and a histidine ligand of each T3. The additional pathway involving a less efficient hydrogen bond (light blue, between the carbonyl of the cysteine and the NH of a T3 histidine ligand, the H is not shown) uses a total of only 10 bonds. Additional O atoms bound to the TNC are also omitted.


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Biochemistry

Figures Figure 1

Figure 2A

Figure 2B


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Figure 3

1000/T


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Biochemistry

Figure 4

For


Biochemistry

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For Table of Contents Only (different versions due to difficulty in upload)


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Intramolecular Electron Transfer in the Bacterial Two-Domain

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