The Active-Site Histidine-10 of Enterococcal NADH Peroxidase Is Not Essential for Catalytic Activity † Edward J. Crane, III, ‡ Derek Parsonage, ‡ and Al Claiborne* Department of Biochemistry, Wake Forest UniVersity Medical Center, Winston-Salem, North Carolina 27157 ReceiVed October 2, 1995; ReVised Manuscript ReceiVed December 21, 1995 X ABSTRACT: In order to test the proposal [Stehle, T., Claiborne, A., & Schulz, G. E. (1993) Eur. J. Biochem. 211, 221-226] that the active-site His10 of NADH peroxidase functions as an essential acid-base catalyst, we have analyzed mutants in which this residue has been replaced by Gln or Ala. The k cat values for both H10Q and H10A peroxidases, and the pH profile for k cat with H10Q, are very similar to those observed with wild-type peroxidase. Both mutants, however, exhibit K m (H 2 O 2 ) values much higher (50-70-fold) than that for wild-type enzyme, and stopped-flow analysis of the H 2 O 2 reactivity of two-electron reduced H10Q demonstrates that this difference is due to a 150-fold decrease in the second-order rate constant for this reaction with the mutant. Stopped-flow analyses also confirm that reduction of the enzyme by NADH is essentially unaffected by His10 replacement and remains largely rate-limiting in turnover; the formation of an E‚NADH intermediate in the conversion of EfEH 2 is confirmed by diode-array spectral analyses with H10A. Both H10Q and H10A mutants, in their oxidized E(FAD, Cys42-sulfenic acid) forms, exhibit enhanced long-wavelength absorbance bands (λ max ) 650 nm and 550 nm, respectively), which most likely reflect perturbations in a charge-transfer interaction between the Cys42-sulfenic acid and FAD. Combined with the 50-fold increase in the second-order rate constant for H 2 O 2 inactivation (via Cys42- sulfenic acid oxidation) of the H10Q mutant, these observations support the proposal that His10 functions in part to stabilize the unusual Cys42-sulfenic acid redox center within the active-site environment. Although structurally related to flavoprotein disulfide reductases such as glutathione reductase (GR; 1 Williams, 1992), the NADH peroxidase from Enterococcus faecalis 10C1 is unique in that it utilizes the Cys42 thiol/sulfenic acid (-SH/-SOH) redox couple in the heterolytic cleavage of the peroxide bond (Claiborne et al., 1993, 1994). Recent studies from this laboratory not only have documented the essential role of Cys42 in the catalytic redox cycle (Parsonage & Claiborne, 1995) but also have investigated the properties of an L40C mutant which contains an active-site Cys40- Cys42 disulfide (Miller et al., 1995). The kinetic mechanism of the wild-type peroxidase has been shown (Crane et al., 1995) to involve (1) NADH reduction of E(FAD, Cys42- SOH)fEH 2 (FAD, Cys42-SH) in an initial priming step; (2) rapid binding of NADH to EH 2 ; (3) reduction of H 2 O 2 by the Cys42-thiolate, yielding E‚NADH; and (4) rate-limiting hydride transfer from bound NADH, regenerating EH 2 . No discrete FADH 2 intermediate has been observed, however, and the precise details of Cys42-SOH reduction have not been elucidated. The pH profile for k cat reveals a relatively acidic optimum of 5.0-5.5 with an apparent limiting pK a of 6.9, consistent with the requirement for a proton in the rate- limiting hydride transfer step. The active-site base in Escherichia coli GR is His439 (Greer & Perham, 1986; Mittl & Schulz, 1994), which is located near the C-terminus of the complementary subunit and interacts with the Cys42-Cys47 redox-active disulfide of the reference subunit. Recent studies of the H439A mutant (Rietveld et al., 1994) have shown that this protein has only 0.3% residual activity; specifically, the limiting rate of GSSG reduction by the corresponding EH 2 species is decreased by approximately 4800-fold. Rietveld et al. (1994) have suggested that His439 serves, in the oxidative half- reaction, to protonate the nascent thiolate of the first GSH product (pK a ) 9.7; Dawson et al., 1986), thus preventing the reverse reaction. In contrast to the GR active-site histidine, His10 of the NADH peroxidase is located near the N-terminus of the R1 helix within the FAD-binding R- fold and interacts with Cys42 (observed in the crystal structure as the non-native Cys42-sulfonic acid; Cys42- SO 3 H) of the same subunit within the tetrameric enzyme (Stehle et al., 1991, 1993). Among the six partial and complete NADH peroxidase and NADH oxidase sequences available (Ross & Claiborne, 1991, 1992; Miller et al., 1990; Matsumoto et al., 1995; Fraser et al., 1995; GenBank Accession Number U19610), His10 is absolutely conserved. Stehle et al. (1993), on the basis of the active-site structure of the NADH peroxidase E(Cys42-SO 3 H) complex with NADH, proposed that His10 protonates the nascent hydrox- ide ion (pK a ) 15.7; March, 1985) formed on reduction of H 2 O 2 by the EH 2 Cys42-thiolate. This proposal has been brought into question more recently, however, by the † This work was supported by National Institutes of Health Grant GM-35394. E.J.C. is the recipient of National Research Service Award GM-16274. * To whom correspondence should be addressed at the Department of Biochemistry, Wake Forest University Medical Center, Medical Center Blvd., Winston-Salem, NC 27157. Telephone: (910) 716-3914. Fax: (910) 716-7671. URL: http://invader.bgsm.wfu.edu:80/. ‡ These authors contributed equally to this work. X Abstract published in AdVance ACS Abstracts, February 1, 1996. 1 The abbreviations: GR, glutathione reductase; E, oxidized enzyme; EH2, two-electron reduced enzyme; EH4, four-electron reduced enzyme; Cys42-SOH, Cys42-sulfenic acid; Cys42-SO3H, Cys42-sulfonic acid derivative observed in peroxide-inactivated peroxidase; IPTG, isopropyl -D-thiogalactopyranoside; E′ 0 , midpoint redox potential at pH 7.0; E1, midpoint potential for the redox couple EH2/EH4; E2, midpoint potential for the redox couple E/EH2. 2380 Biochemistry 1996, 35, 2380-2387 0006-2960/96/0435-2380$12.00/0 © 1996 American Chemical Society