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Nitric oxide synthase (NOS) is a cytochrome P450-type mono-oxygenase that catalyzes the oxidation of l-arginine (Arg) to nitric oxide (NO) through a reaction intermediate N-hydroxy-l-arginine (NHA). The mechanism underlying the reaction catalyzed by NOS from Deinococcus radiodurans was investigated using pulse radiolysis. Radiolytically generated hydrated electrons reduced the heme iron of NOS within 2 μs. Subsequently, ferrous heme reacted with O2 to form a ferrous-dioxygen intermediate with a second-order rate constant of 2.8 × 108 M–1 s–1. In the tetrahydrofolate (H4F)-bound enzyme, the ferrous-dioxygen intermediate was found to decay an another intermediate with a first-order rate constant of 2.2 × 103 s–1. The spectrum of the intermediate featured an absorption maximum at 440 nm and an absorption minimum at 390 nm. In the absence of H4F, this step did not proceed, suggesting that H4F was reduced with the ferrous-dioxygen intermediate to form a second intermediate. The intermediate further converted t...

In Escherichia coli, the [2Fe-2S] transcriptional factor, SoxR, functions as a sensor of oxidative stress. The transcriptional activity in SoxR is regulated by the reversible oxidation and reduction of [2Fe-2S] clusters. We previously proposed that superoxide (O2•–) has a direct role as a signal for E. coli SoxR and that the sensitivity of the E. coli SoxR response to O2•– is 10-fold higher than that of Pseudomonas aeruginosa SoxR. The difference between the two homologues reflects interspecies differences in the regulatory role of O2•– activation. To investigate the determinants of SoxR’s sensitivity to O2•–, we substituted several amino acids that are not conserved among enteric bacteria SoxR homologues and investigated the interaction of SoxR with O2•– using pulse radiolysis. The substitution of E. coli SoxR Lys residues 89 and 92 with Ala residues (K89AK92A), located close to [2Fe-2S] clusters, dramatically affected this protein’s reaction with O2•–. The second-order rate constant of the reaction was ...

The iron response regulator (Irr) protein from Bradyrhizobium japonicum mediates iron-dependent regulation of heme biosynthesis. Irr degrades in response to heme availability through a process that involves the binding of heme to Cys-29 in the heme regulatory motif (HRM) in the presence of molecular oxygen. In this work, we assessed the dynamics of one-electron reduction of heme-bound Irr by monitoring the formation of transient intermediates by pulse radiolysis. Hydrated electrons generated by pulse radiolysis reduced heme iron-bound Irr, facilitating the binding of molecular oxygen to the heme iron in Irr through an initial intermediate with an absorption maximum at 420 nm. This initial intermediate was converted to a secondary intermediate with an absorption maximum at 425 nm, with a first-order rate constant of 1.0 × 10(4) s(-1). The Cys-29 → Ala (C29A) mutant of Irr, on the other hand, did not undergo the secondary phase, implying that ligand exchange of Cys-29 for another ligand takes place during the process. Spectral changes during the reduction of the heme-bound Irr revealed that binding of CO to ferrous heme consisted of two phases with kon values of 1.3 × 10(5) and 2.5 × 10(4) M(-1) s(-1), a finding consistent with the presence of two distinct hemes in Irr. In aerobic solutions, by contrast, oxidation of the ferrous heme to the ferric form was found to be a two-phase process. The C29A mutant was similarly oxidized, but this occurred as a single-phase process. We speculate that a reactive oxygen species essential for degradation of the protein is generated during the oxidation process.

The [2Fe-2S] transcriptional factor SoxR, a member of the MerR family, functions as a sensor of oxidative stress in Escherichia coli. The transcriptional activity of SoxR is regulated by the reversible oxidation and reduction of [2Fe-2S] clusters. Electrochemistry measurements on DNA-modified electrodes have shown a dramatic shift in the reduction potential of SoxR from -290 to +200 mV with the promoter DNA-bound [ Gorodetsky , A. A. , Dietrich , L. E. P. , Lee , P. E. , Demple , B. , , Newman , D. K. , and Barton , J. K. ( 2008 ) DNA binding shifts the reduction potential of the transcription factor SoxR , Proc. Natl. Acad. Sci. U.S.A. 105 , 3684 - 3689 ]. To determine the change of the SoxR reduction potential using the new condition, the one-electron oxidation-reduction properties of [2Fe-2S] cluster in SoxR were investigated in the absence and presence of the DNA. The [2Fe-2S] cluster of SoxR was completely reduced by nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CRP) in the presence of a NADPH generating system (glucose 6-dehydrogenase and glucose-6 phosphate), indicating that CRP can serve as an NADPH-dependent electron carrier for SoxR. The reduction potential of SoxR was measured from equilibrium data coupled with NADPH and CRP in the presence of electron mediators. The reduction potentials of DNA-bound and DNA-free states of SoxR were -320 and -293 mV versus NHE (normal hydrogen electrode), respectively. These results indicate that DNA binding causes a moderate shift in the reduction potential of SoxR.

YddV from Escherichia coli (Ec) is a novel globin-coupled heme-based oxygen sensor protein displaying diguanylate cyclase activity in response to oxygen availability. In this study, we quantified the turnover numbers of the active [Fe(III), 0.066 min(-1); Fe(II)-O(2) and Fe(II)-CO, 0.022 min(-1)] [Fe(III), Fe(III)-protoporphyrin IX complex; Fe(II), Fe(II)-protoporphyrin IX complex] and inactive forms [Fe(II) and Fe(II)-NO,0.01 min(-1)] of YddV for the first time. Our data indicate that the YddV reaction is the rate-determining step for two consecutive reactions coupled with phosphodiesterase Ec DOS activity on cyclic di-GMP (c-di-GMP) [turnover number of Ec DOS-Fe(II)-O(2), 61 min(-1)]. Thus, O(2) binding and the heme redox switch of YddV appear to be critical factors in the regulation of c-di-GMP homeostasis. The redox potential and autoxidation rate of heme of the isolated heme domain of YddV (YddV-heme) were determined to be -17 mV versus the standard hydrogen electrode and 0.0076 min(-1), respectively. The Fe(II) complexes of Y43A and Y43L mutant proteins (residues at the heme distal side of the isolated heme-bound globin domain of YddV) exhibited very low O(2) affinities, and thus, their Fe(II)-O(2) complexes were not detected on the spectra. The O(2) dissociation rate constant of the Y43W protein was150 s(-1), which is significantly larger than that of the wild-type protein (22 s(-1)). The autoxidation rate constants of the Y43F and Y43W mutant proteins were 0.069 and 0.12 min(-1), respectively, which are also markedly higher than that of the wild-type protein. The resonance Raman frequencies representing ν(Fe-O(2)) (559 cm(-1)) of the Fe(II)-O(2) complex and ν(Fe-CO) (505 cm(-1)) of the Fe(II)-CO complex of Y43F differed from those (ν(Fe-O(2)), 565 cm(-1); ν(Fe-CO), 495 cm(-1)) of the wild-type protein, suggesting that Tyr43 forms hydrogen bonds with both O(2) and CO molecules. On the basis of the results, we suggest that Tyr43 located at the heme distal side is important for the O(2) recognition and stability of the Fe(II)-O(2) complex, because the hydroxyl group of the residue appears to interact electrostatically with the O(2) molecule bound to the Fe(II) complex in YddV. Our findings clearly support a role of Tyr in oxygen sensing, and thus modulation of overall conversion from GTP to pGpG via c-di-GMP catalyzed by YddV and Ec DOS, which may be applicable to other globin-coupled oxygen sensor enzymes.

Deprotonation of the adenine (A) base in both mononucleotide and oligonucleotide (ODN) was measured by nanosecond pulse radiolysis. The cation radical (A(+*)) of deoxyadenosine (dA), produced by oxidation with SO(4)(-*), rapidly deprotonated to form the neutral A radical (A(- H)(*)) with a rate constant of 2.0 x 10(7) s(-1) and a pK(a) value of 4.2, as determined by transient spectroscopy. A similar process was observed in experiments performed on a variety of double-stranded ODNs containing adenine x thymine (A x T) base pairs. The transient spectrum of A(+)(*) in an ODN composed of alternating A x T pairs was essentially identical to that of free dA and differed from the spectra of ODNs containing AA and AAA. In contrast, the spectra of A(- H)(*) were not affected by the sequence. These results suggest that the positive charge on A(+)(*) in ODNs is delocalized as the dimer is stabilized by pi-orbital stacking between adjacent A's. The rate constants for deprotonation of A(+)(*) in ODNs containing AA and AAA (0.9-1.1 x 10(7) s(-1)) were a factor of 2 smaller than the rate constants for deprotonation of A(+)(*) in ODNs containing alternating A x T and dA (2.0 x 10(7) s(-1)). This suggests that the formation of a charge resonance stabilized dimer AA(+)(*) in DNA produced a significant barrier to deprotonation.

Cytochromes b(561), a novel class of transmembrane electron transport proteins residing in a large variety of eukaryotic cells, have a number of common structural features including six hydrophobic transmembrane alpha-helices and two heme ligation sites. We found that recombinant Zea mays cytochrome b(561) obtained by a heterologous expression system using yeast Pichia pastoris cells could utilize the ascorbate/mondehydroascorbate radical as a physiological electron donor/acceptor. We found further that a concerted proton/electron transfer mechanism might be operative in Z. mays cytochrome b(561) as well upon the electron acceptance from ascorbate to the cytosolic heme center. The well-conserved Lys(83) residue in a cytosolic loop was found to have a very important role(s) for the binding of ascorbate and the succeeding electron transfer via electrostatic interactions based on the analyses of three site-specific mutants, K83A, K83E, and K83D. Further, unusual behavior of the K83A mutant in pulse radiolysis experiments indicated that Lys(83) might also be responsible for the intramolecular electron transfer to the intravesicular heme. On the other hand, pulse radiolysis experiments on two site-specific mutants, S118A and W122A, for the well-conserved residues in the putative monodehydroascorbate radical binding site showed that their electron transfer activities to the monodehydroascorbate radical were very similar to those of the wild-type protein, indicating that Ser(118) and Trp(122) do not have major roles for the redox events on the intravesicular side.

The dynamics of electron adducts of 2'-deoxynucleotides and oligonucelotides (ODNs) were measured spectroscopically by nanosecond pulse radiolysis. The radical anions of the nucleotides were produced within 10 ns by the reaction of hydrated electrons (e(aq)(-)) and were protonated to form the corresponding neutral radicals. At pH 7.0, the radical anion of deoxythymidine (dT(*-)) was protonated to form the neutral radical dT(H)(*) in the time range of microseconds. The rate constant for the protonation was determined as 1.8 x 10(10) M(-1) s(-1). In contrast, the neutral radical of dC(H)(*) was formed immediately after the pulse, suggesting that the protonation occurs within 10 ns. The transient spectra of excess electrons of the double-stranded ODNs 5'-TAATTTAATAT-3' (AT) and 5'-CGGCCCGGCGC-3' (GC) differed from those of pyrimidine radicals (C and T) and their composite. In contrast, the spectra of the electron adducts of the single-stranded ODNs GC and AT exhibited characteristics of C and T, respectively. These results suggest that, in duplex ODNs, the spectral intermediates of G-C and A-T anions complex were formed. On the microsecond time scale, the subsequent changes in absorbance of the ODN AT had a first-order rate constant of 4 x 10(4) s(-1), reflecting the protonation of T.

The deprotonation of guanine cation radical (G+*) in oligonucleotides (ODNs) was measured spectroscopically by nanosecond pulse radiolysis. The G+* in ODN, produced by oxidation with SO4-*, deprotonates to form the neutral G radical (G(-H)*). In experiments using 5-substituted cytosine-modified ODN, substitution of the cytosine C5 hydrogen by a methyl group increased the rate constant of deprotonation, whereas replacement by bromine decreased the rate constant. Kinetic solvent isotope effects on the kinetics of deoxyguanosine (dG) and ODN duplexes were examined in H2O and D2O. The rate constant of formation of G(-H)* in dG was 1.7-fold larger in H2O than D2O, whereas the rate constant in the ODN duplex was 3.8-fold larger in H2O than D2O. These results suggest that the formation of G(-H)* from G+* in the ODN corresponds to the deprotonation of the oxidized hydrogen-bridged (G+*-C) base pair by a water molecule. The characteristic absorption maxima of G+* around 400 nm were shifted to a longer wavelength in the order of G

The dynamics of one-electron oxidation of guanine (G) base mononucleotide and that in DNA have been investigated by pulse radiolysis. The radical cation (G+*) of deoxyguanosine (dG), produced by oxidation with SO(4)-*, rapidly deprotonates to form the neutral G radical (G(-H)*) with a rate constant of 1.8 x 10(7) s(-1) at pH 7.0, as judged from transient spectroscopy. With experiments using different double-stranded oligonucleotides containing G, GG, and GGG sequences, the absorbance increases at 625 nm, characteristic of formation of the G(-H)*, were found to consist of two phases. The rate constants of the faster ( approximately 1.3 x 10(7) s(-1)) and slower phases ( approximately 3.0 x 10(6) s(-1)) were similar for the different oligonucleotides. On the other hand, in the oligonucleotide containing G located at the 5'- and 3'-terminal positions, only the faster phase was seen. These results suggest that the lifetime of the radical cation of the G:C base pair (GC+*), depending on its location in the DNA chain, is longer than that of free dG. In addition, the absorption spectral intermediates showed that hole transport to a specific G site within a 12-13mer double-stranded oligonucleotide is complete within 50 ns; that is, the rate of hole transport over 20 A is10(7) s(-1).

We have previously prepared beta-lactoglobulin (beta-LG)-carboxymethyl dextran (CMD) conjugates with water-soluble carbodiimide and achieved reduced immunogenicity of beta-LG. In the present study, to elucidate the mechanism for the reduced immunogenicity of beta-LG, we investigated changes in the T cell response to beta-LG after conjugation with CMDs differing in molecular weight (about 40 and 162 kDa). Lymph node cells from BALB/c, C3H/He, and C57BL/6 mice that had been immunized with beta-LG or the conjugates were stimulated with beta-LG, and the in vivo T cell response was then evaluated by BrdU (5-bromo-2'-deoxyuridine) ELISA as the ex vivo proliferative response. T cells from the conjugate-immunized mice showed a lower proliferative response than those from the beta-LG-immunized mice. T cell epitope scanning, using synthesized peptides, showed that the T cell epitope profiles of the conjugates were similar to those of beta-LG, whereas the proliferative response to each epitope was reduced. These results indicate that the lower in vivo T cell response with the conjugates was not due to induction of conjugate-specific T cells, but due to a decrease in the number of beta-LG-specific T cells. After the lymph node cells from beta-LG-immunized mice had been stimulated with beta-LG or the conjugates, the efficiency of the antigen presentation of the conjugate to beta-LG-specific T cells was evaluated by BrdU ELISA as the in vitro proliferative response. The antigen presentation of beta-LG to the T cells was reduced by conjugation with CMD. In addition, conjugation with CMD enhanced the resistance of beta-LG to cathepsin B and cathepsin D, which suggest that conjugation with CMD inhibited the degradation of beta-LG by proteases in APC and led to suppression of the generation of antigenic peptides including T cell epitopes from beta-LG. It is therefore considered that the suppressive effect on the generation of T cell epitopes reduced the antigen presentation of the conjugates and that this reduction led to a decrease in the number of beta-LG-specific T cells in vivo. As a result, the decreased help to B cells by T cells would have reduced the antibody response to beta-LG. We conclude that suppression of the generation of T cell epitopes by conjugation with CMD is important to the mechanism for the reduced immunogenicity of beta-LG.

To reduce the immunogenicity of beta-lactoglobulin (beta-LG), two beta-LG-carboxymethyl dextran (CMD) conjugates (Conj. 40 and Conj. 162) were prepared by using water-soluble carbodiimide (EDC). The molar ratios of beta-LG to CMD in Conj. 40 and Conj. 162 were 8:1 and 7:1, respectively. Each conjugate maintained approximately 50% of the retinol binding activity of beta-LG. Structural analyses by intrinsic fluorescence, CD spectra, and ELISA with monoclonal antibodies indicated that the surface of beta-LG in each conjugate was covered by CMD without great disruption of native conformation. By conjugation with CMD, the antibody response to beta-LG was reduced in BALB/c, C3H/He, and C57BL/6 mice, which was eminent in Conj. 162. The results of B cell epitope scanning using overlapping synthesized peptides showed that the linear epitope profiles of the conjugates were similar to those of beta-LG, whereas the antibody response to each epitope was reduced, which was eminent in Conj. 162. It was concluded that conjugation with CMD of higher molecular weight is effective in reducing the immunogenicity of beta-LG and that masking of epitopes by CMD is responsible for the reduced immunogenicity.

To elucidate a unique mechanism for the quinol oxidation in the Escherichia coli cytochrome bo, we applied pulse radiolysis technique to the wild-type enzyme with or without a single bound ubiquinone-8 at the high-affinity quinone binding site (Q(H)), using N-methylnicotinamide (NMA) as an electron mediator. With the ubiquinone bound enzyme, the reduction of the oxidase occurred in two phases as judged from kinetic difference spectra. In the faster phase, the transient species with an absorption maximum at 440 nm, a characteristic of the formation of ubisemiquinone anion radical, appeared within 10 micros after pulse radiolysis. In the slower phase, a decrease of absorption at 440 nm was accompanied by an increase of absorption at 428 and 561 nm, characteristic of the reduced form. In contrast, with the bound ubiquinone-8-free wild-type enzyme, NMA radicals directly reduced hemes b and o, though the reduction yield was low. These results indicate that a pathway for an intramolecular electron transfer from ubisemiquinone anion radical at the Q(H) site to heme b exists in cytochrome bo. The first-order rate constant of this process was calculated to be 1.5 x 10(3) s(-1) and is comparable to a turnover rate for ubiquinol-1. The rate constant for the intramolecular electron transfer decreased considerably with increasing pH, though the yields of the formation of ubisemiquinone anion radical and the subsequent reduction of the hemes were not affected. The pH profile was tightly linked to the stability of the bound ubisemiquinone in cytochrome bo [Ingledew, W. J., Ohnishi, T., and Salerno, J. C. (1995) Eur. J. Biochem. 227, 903-908], indicating that electron transfer from the bound ubisemiquinone at the Q(H) site to the hemes slows down at the alkaline pH where the bound ubisemiquinone can be stabilized. These findings are consistent with our previous proposal that the bound ubiquinone at the Q(H) site mediates electron transfer from the low-affinity quinol oxidation site in subunit II to low-spin heme b in subunit I.

Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups and transports electron equivalents across the vesicle membranes to convert intravesicular monodehydroascorbate radical to ascorbate. To elucidate the mechanism of the transmembrane electron transfer, effects of the treatment of purified cytochrome b(561) with diethyl pyrocarbonate, a reagent specific for histidyl residues, were examined. We found that when ascorbate was added to the oxidized form of diethyl pyrocarbonate-treated cytochrome b(561), less than half of the heme iron was reduced but with a very slow rate. In contrast, radiolytically generated monodehydroascorbate radical was oxidized rapidly by the reduced form of diethyl pyrocarbonate-modified cytochrome b(561), as observed for untreated cytochrome b(561). These results indicate that the heme center specific for the electron acceptance from ascorbate was perturbed by the modification of amino acid residues nearby. We identified the major modification sites by mass spectrometry as Lys85, His88, and His161, all of which are fully conserved and located on the extravesicular side of cytochrome b(561) in the membranes. We suggest that specific N-carbethoxylation of the histidyl ligands of the heme b at extravesicular side abolishes the electron-accepting ability from ascorbate.

Cytochrome bd is a two-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and binds hemes b558, b595, and d as the redox metal centers. Taking advantage of spectroscopic properties of three hemes which exhibit distinct absorption peaks, we investigated electron transfer within the enzyme by the technique of pulse radiolysis. Reduction of the hemes in the air-oxidized, resting-state enzyme, where heme d exists in mainly an oxygenated form and partially an oxoferryl and a ferric low-spin forms, occurred in two phases. In the faster phase, radiolytically generated N-methylnicotinamide radicals simultaneously reduced the ferric hemes b558 and b595 with a second-order rate constant of 3 x 10(8) M-1 s-1, suggesting that a rapid equilibrium occurs for electron transfer between two b-type hemes long before 10 micros. In the slower phase, an intramolecular electron transfer from heme b to the oxoferryl and the ferric heme d occurred with the first-order rate constant of 4.2-5.6 x 10(2) s-1. In contrast, the oxygenated heme d did not exhibit significant spectral change. Reactions with the fully oxidized and hydrogen peroxide-treated forms demonstrated that the oxidation and/or ligation states of heme d do not affect the heme b reduction. The following intramolecular electron transfer transformed the ferric and oxoferryl forms of heme d to the ferrous and ferric forms, respectively, with the first-order rate constants of 3.4 x 10(3) and 5.9 x 10(2) s-1, respectively.

Electron transfer within cytochrome cd1 from Thiosphaera pantotropha was investigated by the technique of pulse radiolysis. The reduction of the heme centers in this nitrite reductase occurred in two phases as judged from kinetic difference spectra. In the faster phase, radiolytically generated N-methylnicotinamide (NMA) radicals selectively reduced the c-heme of the enzyme. From the absorbance increase at 420 nm, a characteristic of formation of the ferrousc-heme, the second-order rate constant for this electron transfer process was estimated to be 3.8 x 10(9) M-1 s-1 at pH 7.0. In the slower phase, a decrease of absorption around 420 and 550 nm, corresponding to a reoxidation of the c-heme, was accompanied by an increase of absorption around 460 and 640 nm, characteristic of formation of the reduced d1-heme. This indicated that an intramolecular electron transfer from the c-heme to the d1-heme occurred. The first-order rate constant of this process was calculated to be 1.4 x 10(3) s-1 at pH 7.0 and was independent of the enzyme concentration. In the presence of nitrite the interheme electron transfer rate was not affected, but on a time scale of seconds a new species associated with the d1-heme, having an absorption maximum at 640 nm, was detected and is proposed to reflect ligand binding to this heme. These results suggest the role of the c-heme as the electron acceptor site in cytochrome cd1 and in mediating the electron transfer to the catalytic site of the enzyme. Moreover, the fast interheme electron transfer rate argues against this process being the rate determining step in catalysis.

The kinetic analysis of the recombination of NO with some ferric hemoproteins was performed by the use of flash photolysis and stopped-flow methods. The rate constants for recombination of NO with ferrimyoglobin obtained by the two methods were identical with each other in the whole pH range. The rate constants decreased with an increase in pH, giving a pK value of 8.5 (cf. 5.2 x 10(4) M-1 s-1 at pH 6 and 1.3 x 10(4) M-1 s-1 at pH 10). The kinetic difference spectra of NO-ferrimyoglobin at 1 ms after flash were identical with the difference spectra of NO-ferrimyoglobin minus ferrimyoglobin at corresponding pHs. Unlike NO-ferrimyoglobin, NO-ferrihorseradish peroxidase gave different kinetics of NO binding for the two methods. Between pH 9.4 and 11.8, the velocity of NO recombination with the enzyme measured by flash photolysis remained constant, but that by the flow method decreased with increasing pH. Below pH 9.4, both methods gave an identical value of 1.9 x 10(5) M-1 s-1. The kinetic difference spectra showed that the acid form, but not the alkaline form, appeared first upon photolysis of NO-ferrihorseradish peroxidase even at alkaline pH. The acid form of peroxidase isoenzyme C re-formed the NO complex, while that of peroxidase isoenzyme A produced a mixture of the NO complex and the alkaline form. The data obtained here were compatible with the assumption that the formation of the alkaline form of the enzymes is the coordination of OH- at the sixth position, which is vacant at acidic pHs.