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The decaheme enzyme cytochrome

Cytochrome c nitrite reductase (ccNiR) is a periplasmic, decaheme homodimeric enzyme that catalyzes the six-electron reduction of nitrite to ammonia. Under standard assay conditions catalysis proceeds without detected intermediates, and it has been assumed that this is also true in vivo. However, this report demonstrates that it is possible to trap a putative intermediate by controlling the electrochemical potential at which reduction takes place. UV/vis spectropotentiometry showed that nitrite-loaded Shewanella oneidensis ccNiR is reduced in a concerted two-electron step to generate an {FeNO}7 moiety at the active site, with an associated midpoint potential of +246 mV vs SHE at pH 7. By contrast, cyanide-bound active site reduction is a one-electron process with a midpoint potential of +20 mV, and without a strong-field ligand the active site midpoint potential shifts 70 mV lower still. EPR analysis subsequently revealed that the {FeNO}7 moiety possesses an unusual spectral signature, different from those normally observed for {FeNO}7 hemes, that may indicate magnetic interaction of the active site with nearby hemes. Protein film voltammetry experiments previously showed that catalytic nitrite reduction to ammonia by S. oneidensis ccNiR requires an applied potential of at least -120 mV, well below the midpoint potential for {FeNO}7 formation. Thus, it appears that an {FeNO}7 active site is a catalytic intermediate in the ccNiR-mediated reduction of nitrite to ammonia, whose degree of accumulation depends exclusively on the applied potential. At low potentials the species is rapidly reduced and does not accumulate, while at higher potentials it is trapped, thus preventing catalytic ammonia formation.

Attempts to selectively oxidize mustard gas [(ClCH2CH2)2S, abbreviated as BCES] to the non-toxic sulfoxide using a trans-Ru(TMP)(O)2/O2 catalyst (TMP = porphyrin dianion of 5,10,15,20-tetramesitylporphyrin) have led to isolation and characterization, including an X-ray structure, of trans-Ru(TMP)(BCES)2, the first such report of a metal-mustard gas complex.

A re-investigation of the interaction with NO of the small tetraheme protein cytochrome c554 (C554) from Nitrosomonas europaea has shown that the 5-coordinate heme II of the two- or four-electron-reduced protein will nitrosylate reversibly. The process is first order in C554, first order in NO, and second-order overall. The rate constant for NO binding to the heme is 3000 ± 140 M-1s-1, while that for dissociation is 0.034 ± 0.009 s-1; the degree of protein reduction does not appear to significantly influence the nitrosylation rate. In contrast to a previous report (Upadhyay AK, et al. J Am Chem Soc 128:4330, 2006), this study found no evidence of C554-catalyzed NO reduction, either with [Formula: see text] or with [Formula: see text] Some sub-stoichiometric oxidation of the lowest potential heme IV was detected when [Formula: see text] was exposed to an excess of NO, but this is believed to arise from partial intramolecular electron transfer that generates {Fe(NO)}8 at heme II. The vacant heme II coordination site of C554 is crowded by three non-bonding hydrophobic amino acids. After replacing one of these (Phe156) with the smaller alanine, the nitrosylation rate for F156A2- and F156A4- was about 400× faster than for the wild type, though the rate of the reverse denitrosylation process was almost unchanged. Unlike in the wild-type C554, the 6-coordinate low-spin hemes of F156A4- oxidized over the course of several minutes after exposure to NO. Concomitant formation of N2O could explain this heme oxidation, though alternative explanations are equally plausible given the available data.

Cytochrome

The previously reported nitric oxide precursor [Mn(PaPy2Q)NO]ClO4 (1), where (PaPy2QH) is N,N-bis(2-pyridylmethyl)-amine-N-ethyl-2-quinoline-2-carboxamide, was used to investigate the interaction between NO and the protein truncated hemoglobin N (trHbN) from the pathogen Mycobacterium tuberculosis. Oxy-trHbN is exceptionally efficient at converting NO to nitrate, with a reported rate constant of 7.45 × 10(8) M(-1) s(-1) [Ouellet, H., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5902] compared to 4 × 10(7) M(-1) s(-1) for oxy-myoglobin [Eich, R. F., et al. (1996) Biochemistry 35, 6976]. This work analyzed the NO dioxygenation kinetics of wild type oxy-trHbN and a set of variants, as well as the nitrosylation kinetics for the reduced (red-trHbN) forms of these proteins. The NO dioxygenation reaction was remarkably insensitive to mutations, even within the active site, while nitrosylation was somewhat more sensitive. Curiously, the most profound change to the rate constant for nitrosylation was effected by deletion of a 12-amino acid dangling N-terminal sequence. The deletion mutant exhibited first-order kinetics with respect to NO but was zero-order with respect to protein concentration; by contrast, all other variants exhibited second-order rate constants of >10(8) M(-1) s(-1). trHbN boasts an extensive tunnel system that connects the protein exterior with the active site, which is likely the main contributor to the protein's impressive NO dioxygenation efficiency. The results herein suggest that N-terminal deletion abolishes a large scale conformational motion, in the absence of which NO can still readily enter the tunnel system but is then prevented from binding to the heme for an extended period of time.

A re-investigation of the interaction with NO of the small tetraheme protein cytochrome c

Multielectron multiproton reactions play an important role in both biological systems and chemical reactions involved in energy storage and manipulation. A key strategy employed by nature in achieving such complex chemistry is the use of proton-coupled redox steps. Cytochrome c nitrite reductase (ccNiR) catalyzes the six-electron seven-proton reduction of nitrite to ammonia. While a catalytic mechanism for ccNiR has been proposed on the basis of studies combining computation and crystallography, there have been few studies directly addressing the nature of the proton-coupled events that are predicted to occur along the nitrite reduction pathway. Here we use protein film voltammetry to directly interrogate the proton-coupled steps that occur during nitrite reduction by ccNiR. We find that conversion of nitrite to ammonia by ccNiR adsorbed to graphite electrodes is defined by two distinct phases; one is proton-coupled, and the other is not. Mutation of key active site residues (H257, R103, and Y206) modulates these phases and specifically alters the properties of the detected proton-dependent step but does not inhibit the ability of ccNiR to conduct the full reduction of nitrite to ammonia. We conclude that the active site residues examined are responsible for tuning the protonation steps that occur during catalysis, likely through an extensive hydrogen bonding network, but are not necessarily required for the reaction to proceed. These results provide important insight into how enzymes can specifically tune proton- and electron transfer steps to achieve high turnover numbers in a physiological pH range.

The reaction of nitric oxide with oxy-myoglobin (oxyMb) to form ferric myoglobin (metMb) and nitrate, and the metMb-catalyzed isomerization of peroxynitrite to nitrate, have long been assumed to proceed via the same iron-bound peroxynitrite intermediate (metMb(OONO)). More recent research showed that the metMb-catalyzed isomerization of peroxynitrite to nitrate produces detectable amounts of nitrogen dioxide and ferryl myoglobin (ferrylMb). This suggests a mechanism in which the peroxynitrite binds to the metMb, ferrylMb is transiently generated by dissociation of NO2, and nitrate is formed when the NO2 nitrogen attacks the ferrylMb oxo ligand. The presence of free NO2 and ferrylMb products reveals that small amounts of NO2 escape from myoglobin's interior before recombination can occur. Free NO2 and ferrylMb should also be generated in the reaction of oxyMb with NO, if the common intermediate metMb(OONO) is formed. However, this report presents a series of time-resolved UV/vis spectroscopy experiments in which no ferrylMb was detected when oxyMb and NO reacted. The sensitivity of the methodology is such that as little as 10% of the ferrylMb predicted from the experiments with metMb and peroxynitrite should have been detectable. These results lead to the conclusion that the oxyMb + NO and metMb + ONOO(-) reactions do not proceed via a common intermediate as previously thought. The conclusion has significant implications for researchers that propose a possible role of oxyMb in intracellular NO regulation, because it means that toxic NO2 and ferrylMb are not generated during NO oxidation by this species.

Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, carries out the six-electron reduction of nitrite and the two-electron reduction of hydroxylamine. Protein film voltammetry (PFV) has been used to study the cytochrome c nitrite reductase from Escherichia coli (ecNrfA) previously, revealing catalytic reduction of both nitrite and hydroxylamine substrates by ecNrfA adsorbed to a graphite electrode that is characterized by ‘boosts’ and attenuations in activity depending on the applied potential. Here, we use PFV to investigate the catalytic properties of soNrfA during both nitrite and hydroxylamine turnover and compare those properties to ecNrfA. Distinct differences in both the electrochemical and kinetic characteristics of soNrfA are observed, e.g., all detected electron transfer steps are one-electron in nature, contrary to what has been observed in ecNrfA (Angove, H. C., Cole, J. A., Richardson, D. J., and Butt, J. N. (2002) Protein film voltammetry reveals distinctive fingerprints of nitrite and hydroxylamine reduction by a cytochrome C nitrite reductase, J Biol Chem 277, 23374-23381). Additionally, we find evidence of substrate inhibition during nitrite turnover and negative cooperativity during hydroxylamine turnover, neither of which have previously been observed in any cytochrome c nitrite reductase. Collectively these data provide evidence that during catalysis, potential pathways of communication exist between the individual soNrfA monomers comprising the native homodimer.

We present the structures of bovine catalase in its native form and complexed with ammonia and nitric oxide, obtained by X-ray crystallography. Using the NO generator 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, we were able to generate sufficiently high NO concentrations within the catalase crystals that substantial occupation was observed despite a high dissociation rate. Nitric oxide seems to be slightly bent from the heme normal that may indicate some iron(II) character in the formally ferric catalase. Microspectrophotometric investigations inline with the synchrotron X-ray beam reveal photoreduction of the central heme iron. In the cases of the native and ammonia-complexed catalase, reduction is accompanied by a relaxation phase. This is likely not the case for the catalase NO complex. The kinetics of binding of NO to catalase were investigated using NO photolyzed from N,N'-bis(carboxymethyl)-N,N'-dinitroso-p-phenylenediamine using an assay that combines catalase with myoglobin binding kinetics. The off rate is 1.5 s(-1). Implications for catalase function are discussed.

Hydroxylamine oxidoreductase (HAO) from the ammonia-oxidizing bacterium Nitrosomonas europaea normally catalyzes the four-electron oxidation of hydroxylamine to nitrite, which is the second step in ammonia-dependent respiration. Here we show that, in the presence of methyl viologen monocation radical (MV(red)), HAO can catalyze the reduction of nitric oxide to ammonia. The process is analogous to that catalyzed by cytochrome c nitrite reductase, an enzyme found in some bacteria that use nitrite as a terminal electron acceptor during anaerobic respiration. The availability of a reduction pathway to ammonia is an important factor to consider when designing in vitro studies of HAO, and may also have some physiological relevance. The reduction of nitric oxide to ammonia proceeds in two kinetically distinct steps: nitric oxide is first reduced to hydroxylamine, and then hydroxylamine is reduced to ammonia at a tenfold slower rate. The second step was investigated independently in solutions initially containing hydroxylamine, MV(red), and HAO. Both steps show first-order dependence on nitric oxide and HAO concentrations, and zero-order dependence on MV(red) concentration. The rate constants governing each reduction step were found to have values of (4.7 +/- 0.3) x 10(5) and (2.06 +/- 0.04) x 10(4) M(-1) s(-1), respectively. A second reduction pathway, with second-order dependence on nitric oxide, may become available as the concentration of nitric oxide is increased. Such a pathway might lead to production of nitrous oxide. We estimate a maximum value of (1.5 +/- 0.05) x 10(10) M(-2) s(-1) for the rate constant of the alternative pathway, which is small and suggests that the pathway is not physiologically important.

The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site's midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV-vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme's histidine axial ligands was replaced with a methionine.

We report results of continuum electrostatics calculations of the cofactor redox potentials, and of the titratable group pK a values, in hydroxylamine oxidoreductase (HAO). A picture of a sophisticated multicomponent control of electron flow in the protein emerged from the studies. First, we found that neighboring heme cofactors strongly interact electrostatically, with energies of 50-100 mV. Thus, cofactor redox potentials depend on the oxidation state of other cofactors, and cofactor redox potentials in the active (partially oxidized) enzyme differ substantially from the values obtained in electrochemical redox titration experiments. We found that, together, solvent-exposed heme 1 (having a large negative redox potential) and heme 2 (having a large positive redox potential) form a lock for electrons generated during the oxidation reaction The attachment of HAO's physiological electron transfer partner cytochrome c 5 5 4 results in a positive shift in the redox potential of heme 1, and "opens the electron gate". Electrons generated as a result of hydroxylamine oxidation travel to heme 3 and heme 8, which have redox potentials close to 0 mV versus NHE (this result is in partial disagreement with an existing experimental redox potential assignment). The closeness of hemes 3 and 8 from different enzyme subunits allows redistribution of the four electrons generated as a result of hydroxylamine oxidation, among the three enzyme subunits. For the multielectron oxidation process to be maximally efficient, the redox potentials of the electron-accepting cofactors should be roughly equal, and electrostatic interactions between extra electrons on these cofactors should be minimal. The redox potential assignments presented in the paper satisfy this general rule.

When irradiated with ∼10 ns laser pulses of 308 nm light, N,N′-bis-(carboxymethyl)-N,N′-dinitroso-1,4-phenylenediamine (1) fragments in less than a μs to give NO and the denitrosylated radical of 1 (2). Species 1 has great potential as a research tool because the photo-generated NO can subsequently be used to probe fast reactions of biochemical interest. This study focuses on the properties of the reactive intermediate 2, which must be known before 1 can confidently be used in more complex investigations. Experiments in which 1 was irradiated in the presence of myoglobin (Mb) or ferrocytochrome c revealed that 2 can rapidly oxidize either species. For myoglobin, the rate constant kox was measured as (5.5±0.5)×107 M−1 s−1. Experiments in which large amounts of 2 were photo-generated in the presence of free NO revealed that 2 probably recombines with NO to produce metastable isomers of 1, which have lifetimes of ∼20 ms and liberate additional NO as they decay. Previously, 2 was only known to either rapidly recombine with NO to regenerate 1 exclusively, or fragment further to give a second equivalent of NO and the doubly denitrosylated quinoimine derivative of 1 (3). The experiments with myoglobin, which is a chromophoric NO scavenger, also made it easy to quantify the amount of NO generated under various conditions, thus providing indirect information about the reactions of 2. On the basis of these experiments, the value of the rate constant for fragmentation of 2 into 3 and NO (kd) was revised from the previously published 500–2600 s−1. The NO scavenger experiments also suggest that the metastable isomers of 1, generated by recombination of 2 and NO, liberate additional equivalents of NO as they decay.

Irradiation of N , N ′-bis-(carboxymethyl)- N , N ′-dinitroso-1,4-phenylenediamine ( 1 ) with ultraviolet light ( λ =308 nm) was previously shown to induce cleavage of 1 into NO and the N , N ′-bis-(carboxymethyl)- N -nitroso-1,4-phenylenediamine radical ( 2 ). This paper provides a procedure for synthesizing 1 , and a detailed kinetic re-investigation of the reactions that follow ns-pulse laser-flash photolysis of 1 , under conditions that initially produce up to 10 μM of 2 and NO in situ. Under these conditions recombination of 2 with NO, and dissociation of an additional equivalent of NO from 2 , appear to be the dominant pathways leading to the depletion of 2 in the ms that follows the initial photolysis. These reactions had been suggested in the initial study of the photolysis of 1 ; however, in the present study the rate constant k r that governs the recombination of 2 with NO to regenerate 1 was found to have a value of (1.1±0.1)×10 9 M −1 s −1 , while the rate constant k d governing the dissociation of the second equiv of NO from 2 was found to be 500±50 s −1 . Both of these values are significantly different from the values (1.38×10 8 M −1 s −1 and 2.96×10 4 s −1 ) reported in the earlier study. The present analysis also revealed the previously unreported presence of an absorbing species at t ∞ , that might be the doubly denitrosylated quinoimine derivative of 1 ( 3 ), or a charge-transfer complex of 1 and 3 .

The titration of chicken liver sulfite oxidase (SO) with the one-electron reductant Ti(III) citrate, at pH 7.0, results in nearly quantitative selective reduction of the Mo(VI) center to Mo(V), while the b-type heme center remains in the fully oxidized Fe(III) state. The selective reduction of the Mo(VI/V) couple has been established from electronic and EPR spectra. The elec- tronic spectrum of the Fe(III) heme center is essentially unchanged during the titration, and the continuous wave (CW)-EPR spectrum shows the appearance of the well-known Mo(V) signal due to the low pH (lpH) form of SO. Further confirmation of the selective formation of the Mo(V)/Fe(III) form of SO is provided by the � 1:1 ratio of the integrated intensities of the Mo(V) and low- spin Fe(III) EPR signals after addition of one equivalent of Ti(III). The selective generation of the Mo(V)/Fe(III) form of SO is unexpected, considering that previous microcoulometry and flash photolysis investigations have indicated that the Mo(VI/V) and Fe(III/II) couples of SO have similar reduction potentials at pH 7. The nearly quantitative preparation of the one-electron re- duced Mo(V)/Fe(III) form of SO by reduction with Ti(III) has enabled the interaction between these two paramagnetic metalcenters, which are l inked by a flex- ible loop with no secondary structure, to be investigated for the first time by variable-frequency pulsed electron- electron double resonance (ELDOR) spectroscopy. The ELDOR kinetics were obtained from frozen solutions at 4.2 K at severalmicrowave frequencies by pumping on the narrow Mo(V) signaland observing the effect on the Fe(III) primary echo at both higher and lower fre- quencies within the microwave C-band region. The ELDOR data indicate that freezing the solution of one- electron reduced SO produces localized regions where the concentration of SO approaches that in the crystal structure, which results in the interpair interactions be- ing the dominant dipolar interaction. However, thor- ough analysis of the ELDOR decay curves and simulations suggests a distribution of intramolecular Mo ... Fe distances, consistent with the proposalof mul - tiple conformations in solution for the flexible loop that connects the Mo and heme domains of SO.

Cytochrome c nitrite reductase (ccNiR) from Shewanella oneidensis, which catalyzes the six-electron reduction of nitrite to ammonia in vivo, was shown to oxidize hydroxylamine in the presence of large quantities of this substrate, yielding nitrite as the sole free nitrogenous product. UV-visible stopped-flow and rapid-freeze-quench electron paramagnetic resonance data, along with product analysis, showed that the equilibrium between hydroxylamine and nitrite is fairly rapidly established in the presence of high initial concentrations of hydroxylamine, despite said equilibrium lying far to the left. By contrast, reduction of hydroxylamine to ammonia did not occur, even though disproportionation of hydroxylamine to yield both nitrite and ammonia is strongly thermodynamically favored. This suggests a kinetic barrier to the ccNiR-catalyzed reduction of hydroxylamine to ammonia. A mechanism for hydroxylamine reduction is proposed in which the hydroxide group is first protonated and released as water, leaving what is formally an NH2(+) moiety bound at the heme active site. This species could be a metastable intermediate or a transition state but in either case would exist only if it were stabilized by the donation of electrons from the ccNiR heme pool into the empty nitrogen p orbital. In this scenario, ccNiR does not catalyze disproportionation because the electron-donating hydroxylamine does not poise the enzyme at a sufficiently low potential to stabilize the putative dehydrated hydroxylamine; presumably, a stronger reductant is required for this.

A refocused primary electron spin−echo envelope modulation (RP ESEEM) technique and an adjustable frequency S/C-band pulsed EPR spectrometer have been used to produce ESEEM spectra with the lines due to nearby protons being greatly enhanced relative to those due to distant matrix protons. Application of this technique to the high pH (hpH) form of the Mo(V) center of sulfite oxidase has enabled nearby protons to be directly detected for the first time. Simulation of the RP ESEEM spectrum of the hpH form suggests the presence of two nearby protons that have distributed hyperfine interactions (hfi); these protons are ascribed to a MoV−OH group with strong H-bonding interactions to other nearby proton donors or to the presence of a coordinated H2O ligand. The RP ESEEM technique promises to be widely applicable to the investigation of mutant forms of SO with altered Mo centers and paramagnetic centers in other metalloproteins where a nearby proton of interest is often masked by much more numerous distant proto...

The individual rate constants for intramolecular electron transfer (IET) between the Mo(VI)Fe(II) and Mo(V)Fe(III) forms of chicken liver sulfite oxidase (SO) have been determined at a variety of pH values, and at high and low anion concentrations. Large anions such as EDTA do not inhibit IET as dramatically as do small anions such as SO4(2-) and Cl-, which suggests that specific anion binding at the sterically constrained Mo active site is necessary for IET inhibition to occur.IET may require that SO adopt a conformation in which the Mo and Fe centers are held in close proximity by electrostatic interactions between the predominantly positively charged Mo active site, and the negatively charged heme edge. Thus, small anions which can fit into the Mo active site will weaken this electrostatic attraction and disfavor IET. The rate constant for IET from Fe(II) to Mo(VI) decreases with increasing pH, both in the presence and absence of 50 mM SO4(2-) . However, the rate constant for the reverse process exhibits no significant pH dependence in the absence of SO4(2-), and increases with pH in the presence of 50 mM S04(2-). This behavior is consistent with a mechanism in which IET from Mo(V) to Fe(III) is coupled to proton transfer from Mo(V)-OH to OH-, and the reverse IET process is coupled to proton transfer from H2O to Mo(VI) = O. At high concentrations of small anions, direct access of H2O or OH- to the Mo-OH will be blocked, which provides a second possible mechanism for inhibition of IET by such anions. Inhibition by anions is not strictly competitive, however, and Tyr322 may play an important intermediary role in transferring the proton when an anion blocks direct access of H2O or OH- to the Mo-OH. Competing H-bonding interactions of the Mo-OH moiety with Tyr322 and with the anion occupying the active site may also be responsible for the well-known equilibrium between two EPR-distinct forms of SO that is observed for the two-electron reduced enzyme.

Two-pulse and four-pulse electron spin−echo envelope modulation spectroscopy (ESEEMS) at two operational frequencies and two-dimensional hyperfine sublevel correlation spectroscopy (HYSCORES), have been used to probe the MoV coordination environment of sulfite oxidase in H2O and D2O solutions, buffered at pH 9.5 and 7.0 with ∼100 mM Tris-type buffers. At pH 9.5 the ESEEM and HYSCORE results definitively reveal the presence of one solvent-exchangeable D(H) near the MoV center, probably in the form of a Mo−OH(D) moiety. The orientation of this group is not fixed (although it is substantially restricted) and thus gives rise to a distribution of hyperfine interaction (hfi) parameters. The resulting loss of amplitude makes direct observation of a proton-related line using ESEEM impossible. However, such a line is observable in ESEEM spectra of the comparable deuterated enzyme because the narrower distribution of hfi parameters leads to less line broadening of the ESEEM spectra. ESEEM and HYSCORE spectra of sul...

The molybdenum-containing enzyme sulfite oxidase catalyzes the conversion of sulfite to sulfate, the terminal step in the oxidative degradation of cysteine and methionine. Deficiency of this enzyme in humans usually leads to major neurological abnormalities and early death. The crystal structure of chicken liver sulfite oxidase at 1.9 Å resolution reveals that each monomer of the dimeric enzyme consists of three domains. At the active site, the Mo is penta-coordinated by three sulfur ligands, one oxo group, and one water/hydroxo. A sulfate molecule adjacent to the Mo identifies the substrate binding pocket. Four variants associated with sulfite oxidase deficiency have been identified: two mutations are near the sulfate binding site, while the other mutations occur within the domain mediating dimerization.

The molybdenum(V) coordination environment of sulfite oxidase has been investigated by multifrequency ESEEM spectroscopy in approximately 70 mM phosphate buffer at pH = 6.5 in both H(2)O and D(2)O. The FT-ESEEM spectra in H(2)O typically consist of three lines. One of these lines is always close to twice the Larmor frequency of the P atom (2nu(P)) and is assigned to one or more coordinated phosphates, providing the first direct unambiguous detection of such coordination. Extensive simulations of this phosphate signal at the various operational frequencies indicated that the coordinated phosphate group(s) probably does (do) not adopt a fixed orientation, and as a result, a description of the Mo.P hyperfine interaction required the introduction of a distribution of such orientations, with Mo.P distance(s) of 3.2-3.3 Å. The other two lines in the FT-ESEEM spectra in H(2)O, located at nu(H) and 2nu(H), were assigned to matrix protons. In D(2)O buffer two additional lines, assigned to matrix deuterons, were also seen.

The coupling of an Mo VI O 2 →Mo IV O oxygen atom transfer reaction to a one-electron reoxidation of the resulting Mo(IV) center by an Fe(III) porphyrin center has been investigated in an attempt to model the first two redox steps in the catalytic cycle of the enzyme sulfite oxidase. Reaction of LMo VI O 2 Cl (L=hydrotris(3,5-dimethyl-1-pyrazolyl)borate) with excess tri-phenylphosphine in DMF rapidly and cleanly generates LMo IV OCl(DMF). This Mo(IV) species reacts more slowly with Fe III Cl(TTP) (TTP=tetratolylporphyrin) to give LMo V OCl 2 and Fe II (TTP) as the final products. The kinetics of this one-electron redox reaction have been followed by electronic spectroscopy and by 1 H NMR. The reaction is zero-order in Fe(III), first-order in Mo(IV), and independent of added chloride ion. The first-order rate constant determined by these methods ranged from 1.7−4.6×10 −5 s −1 . An inner-sphere (halogen transfer) mechanism is shown to be most consistent with the data.

Hydroxylamine oxidoreductase (HAO) from the autotrophic bacterium Nitrosomonas europaea catalyzes the 4-e- oxidation of NH2-OH to NO2-. The e- are transferred from NH2OH to an unusual 5-coordinate heme known as P460, which is the active site of HAO, and from there to an array of seven c-type hemes. NO., generated by laser flash photolysis of N,N'-bis(carboxymethyl)-N,N'-dinitroso-1,4-phenylenediamine, is found to act as a 1-e- donor to HAO. Most likely NO. binds P460 to yield a [Fe(NO)]6 moiety, which then hydrolyzes to give the reduced enzyme and NO2-. The [Fe(NO)]6 moiety is also a plausible final intermediate in the oxidation of NH2OH.

The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein "small tetraheme c" replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 °C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-A-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).

Hydroxylamine, an important intermediate in ammonia oxidation by ammonia oxidizing bacteria (AOB), is inherently unstable with respect to disproportionation. The process is slow in neutral solutions, but could potentially be catalyzed by enzymes such as the hydroxylamine oxidoreductases, which normally catalyze the oxidation of ammonia to nitrite in the AOB. Disproportionation could be physiologically important to some AOB under microaerobic conditions, and could also confound in vitro analyses if it occurs and is not taken into consideration. This chapter presents methods for detecting ammonia, nitric oxide, nitrite, nitrous oxide, and isotopically labeled dinitrogen, which are the most thermodynamically favored products of hydroxylamine disproportionation.

Hydroxylamine, an important intermediate in ammonia oxidation by ammonia oxidizing bacteria (AOB), is inherently unstable with respect to disproportionation. The process is slow in neutral solutions, but could potentially be catalyzed by enzymes such as the hydroxylamine oxidoreductases, which normally catalyze the oxidation of ammonia to nitrite in the AOB. Disproportionation could be physiologically important to some AOB under microaerobic conditions, and could also confound in vitro analyses if it occurs and is not taken into consideration. This chapter presents methods for detecting ammonia, nitric oxide, nitrite, nitrous oxide, and isotopically labeled dinitrogen, which are the most thermodynamically favored products of hydroxylamine disproportionation.

Hydroxylamine oxidoreductase (HAO) from Nitrosomonas europaea normally catalyzes oxidation of NH(2)OH to NO(2)(-). This paper reports experiments in which HAO was thermodynamically poised to catalyze reduction of NO(2)(-) to NH(4)(+). HAO was found to catalyze the reduction of NO(2)(-) by methyl viologen monocation radical (MV(red)), displaying a hyperbolic dependence on NO(2)(-) concentration, with a k(cat1) of 6.8 ± 0.3 s(-1) and a K(m1) of 7.6 ± 0.9 mM. HAO also catalyzed the reduction of NH(2)OH by MV(red), with a hyperbolic dependence on NH(2)OH concentration, and a k(cat2) of 245 ± 3 s(-1) and a K(m2) of 6.8 ± 0.2 mM (k(cat1) and k(cat2) reflect the maximum number of electrons transferred from MV(red) per second). We had previously demonstrated that HAO catalyzes the reduction of NO by MV(red) to yield first NH(2)OH and then NH(4)(+). Thus, overall, HAO is seen to act like a cytochrome c nitrite reductase, which catalyzes the six-electron reduction of NO(2)(-) to NH(4)(+) by MV(red). In the presence of Ru(NH(3))(2+) (Ru(II)) and Ru(NH(3))(3+) (Ru(III)) at ratios exceeding 200:1, HAO exhibited no detectable Ru(II)-NO(2)(-) oxidoreductase activity, though such activity is thermodynamically favored. On the other hand, HAO could still catalyze the oxidation of NH(2)OH to NO by Ru(III) under these conditions. The oxidative process exhibited a hyperbolic dependence on NH(2)OH concentration, with a k(cat3) of 98 ± 3 s(-1) and a K(m3) of 5.2 ± 0.8 μM. Finally, HAO was found not to catalyze the disproportionation of NH(2)OH, despite the thermodynamic favorability of such a process, and the apparent opportunity presented by the HAO structure. Mechanisms are proposed to explain all the kinetic data.

Upon interaction with anionic phospholipids, particularly mitochondria-specific cardiolipin (CL), cytochrome c (cyt c) loses its tertiary structure and its peroxidase activity dramatically increases. CL induced peroxidase activity of cyt c has been found to be important for selective CL oxidation in cells undergoing programmed death. During apoptosis, the peroxidase activity and the fraction of CL-bound cyt c markedly increases suggesting that CL may act as a switch to regulate cyt c’s mitochondrial functions. Using cyclic voltammetry and equilibrium redox-titrations, we show that the redox potential of cyt c shifts negatively by 350–400 mV upon binding to CL-containing membranes. Consequently, functions of cyt c as an electron transporter and cyt c reduction by Complex III are strongly inhibited. Further, CL/cyt c complexes are not effective in scavenging superoxide anions and are not effectively reduced by ascorbate. Thus, both redox properties and functions of cyt c change upon interaction with CL in the mitochondrial membrane, diminishing cyt c’s electron donor/acceptor role and stimulating its peroxidase activity.

N,N'-Bis(carboxymethyl)-N,N'-dinitroso-1,4-phenylenediamine (1) fragments to release 1 equiv of NO* and the denitrosated radical of 1 (2), when exposed to a approximately 10 ns, 308 nm laser pulse. Species 2 can fragment to give another equivalent of NO* and the doubly denitrosated quinoimine derivative of 1 (3), it can recombine with NO* to give 1 and ring-nitrosated isomers of 1, or in the presence of a reducing agent, 2 can be reduced (to species 4). Photogenerated NO* can be used to probe fast reactions of biochemical interest, making 1 a valuable research tool. This paper focuses on the chemistry of 2, whose reactivity must be well characterized if 1 is to be used to its full potential. [Ru(NH3)6]2+ (RuII) and [Fe(CN)6]4- (FeII) were both shown to reduce 2, with bimolecular rate constants in the diffusion limit. When solutions initially containing 70 microM of RuII, 20 microM myoglobin (Mb) and varying amounts of 1 were irradiated, the only Mb reaction product was nitrosomyoglobin (MbNO). In contrast, in solutions containing only Mb and 1, Mb is converted to both MbNO and oxidized myoglobin (metMb). When FeII was used in place of RuII, Mb was oxidized to metMb, but approximately 100x more slowly than in solutions containing only Mb and 1. This showed that 2 first oxidized FeII to [Fe(CN)6]3- (FeIII), which then oxidized Mb at the slower rate. The ratio metMb/MbNO obtained in the experiments with FeII was 0.6, whereas the ratio predicted from previously known chemistry of 2 was approximately 1 under the experimental conditions. The result is explained if, upon photolysis, 1 first forms a caged encounter complex [2, NO*], which fragments to give 3 and 2 equiv of NO*, without ever releasing free 2 into solution. This hypothesis was further strengthened by analyzing the amount of NO* generated by photolysis of 1 in the absence of added reductant. The original mechanism underestimates the NO* generated, a problem solved by invoking direct release of NO* and 3 from photolysis of 1.

Hydroxylamine-cytochrome c554 oxidoreductase (HAO) catalyzes the 4-e(-) oxidation of NH(2)OH to NO(2)(-) by cytochrome c554. The electrons are transferred from NH(2)OH to a 5-coordinate heme known as P(460), the active site of HAO. From P(460), c-type hemes transport the electrons through the enzyme to a remote solvent-exposed c-heme, where cyt c554 reduction occurs. When 3-60 microM NO* are photogenerated by laser flash photolysis of N,N'-bis-(carboxymethyl)-N,N'-dinitroso-1,4-phenylenediamine, in a solution containing approximately 1 microM HAO prereduced by 3 e(-)/subunit, the HAO c-heme pool is subsequently oxidized by up to 1 e(-)/HAO subunit. The reaction rate for HAO oxidation shows first-order dependence on [HAO], and zero-order dependence on [NO*] (k(obs) = 1250 +/- 150 s(-)(1)). However, the total HAO oxidized shows hyperbolic dependence on [NO*]. We suggest that NO* first binds reversibly to P(460) giving a {Fe(NO)}(6) moiety. Intramolecular electron transfer (IET) from the c-heme pool then reduces P(460) to {Fe(NO)}.(7) The overall binding constant (K) for formation of {Fe(NO)}(7) from free NO* and 3-e(-) reduced HAO was measured at (7.7 +/- 0.6) x10(4) M(-1). This value is larger than that for typical ferriheme proteins ( approximately 10(4) M(-1)), but much smaller than that for the corresponding ferroheme proteins ( approximately 10(11) M(-1)). The final product generated by nitrosylating 3-e(-) reduced HAO is believed to be the same species obtained by adding NH(2)OH to the fully oxidized enzyme. The experiments described herein suggest that when NH(2)OH and HAO first react, only two of the NH(2)OH electrons end up in the c-heme pool. The other two remain at P(460) as part of an {Fe(NO)}(7) moiety. These results are discussed in relation to earlier studies that investigated the effect of putting fully oxidized and fully reduced HAO under 1 atm of NO*.

Exposure to O(2) (or air) of a CH(2)Cl(2), benzene, or toluene solution containing PhCO(2)H and Ru(OEP)(RR'S)(2) (where OEP = the dianion of 2,3,7,8,12,13,17,18-octaethylporphyrin, R = methyl, ethyl, or decyl, and R' = methyl or ethyl), at ambient conditions, results in the selective oxidation of the axial ligand(s) on the metalloporphyrin complex to the corresponding sulfoxide(s). For example, a CD(2)Cl(2) solution of Ru(OEP)(dms)(2) (dms = dimethyl sulfide) and PhCO(2)H, exposed to 1 atm of O(2) at approximately 20 degrees C for 35 h, is oxidized to Ru(OEP)(dmso)(2), and the intermediates Ru(OEP)(dms)(dmso), [Ru(OEP)(dms)(2)][PhCO(2)], and Ru(OEP)(dms)(PhCO(2)) are identified (s implies sulfur-bonded). Mechanisms invoking in situ formation of H(2)O(2), disproportionation of Ru(III) species, and Ru(IV)=O intermediates are proposed for the O(2) oxidation of the thioether ligands. X-ray analysis of Ru(OEP)(Et(2)SO)(2) confirms that the sulfoxides are S-bonded.

Temperature-dependent magnetic circular dichroism (MCD) spectroscopy has been used for the first time to probe the electronic structure of the Mo active site in sulfite oxidase (SO). The enzyme was poised in the catalytically relevant [Mo(V):Fe(II)] state by anaerobic reduction of the enzyme with the natural substrate, sulfite, in the absence of the physiological oxidant cytochrome c. The [Mo(V):Fe(II)] state is of particular importance, as it is proposed to be a catalytic intermediate in the oxidative half reaction, where SO is reoxidized to the resting [Mo(VI):Fe(III)] state by two sequential one-electron transfers to cytochrome c. The MCD spectrum of the enzyme shows no charge transfer transitions below approximately 17000 cm(-1). This has been interpreted to result from (1) a severe reduction in ene-1,2-dithiolate sulfur in-plane and out-of-plane p orbital mixing, (2) a decrease in the dithiolate sulfur out-of-plane p-Mo d(xy) orbital overlap, and (3) an orthogonal orientation between the vertical cysteine sulfur p (perpendicular to the Mo-Scys sigma-bond) and Mo d(xy) orbitals. The spectroscopically determined cysteine sulfur p-Mo d(xy) bonding scheme in the [Mo(V):Fe(II)] state is consistent with the crystallographically determined O-Mo-Scys-C dihedral angle of approximately 90 degrees and precludes a covalent interaction between the vertical cysteine sulfur p orbital and Mo d(xy), effectively decoupling the cysteine from an effective through-bond electron transfer pathway. We have tentatively assigned a 22250 cm(-1) positive C-term feature in the MCD as the cysteine S(sigma)-->Mo d(xy) charge transfer that becomes allowed by a combination of configuration interaction and low-symmetry; however, the orbital overlap is anticipated to be quite small due to the near orthogonality of these orbitals. Therefore, we propose that the primary role of the coordinated cysteine is to decrease the effective nuclear charge on Mo by charge donation to the metal, statically poising the active site at more negative reduction potentials during electron transfer (ET) regeneration. Finally, the results of this study are consistent with the pyranopterin ene-1,2-dithiolate acting to couple the Mo site into efficient superexchange pathways for ET regeneration following oxygen atom transfer to the substrate.

Femtosecond time-resolved absorbance measurements were used to probe the subpicosecond primary events of the photoactive yellow protein (PYP), a 14-kD soluble photoreceptor from Ectothiorhodospira halophila. Previous picosecond absorption studies from our laboratory have revealed the presence of two new early photochemical intermediates in the PYP photocycle, I(0), which appears in

Adducts with MoO 4 2− tetrahedra coordinated to Cr(III) or Co(III) complexes have been synthesized and studied by IR and high resolution 95 Mo NMR spectroscopy. The 95 Mo chemical shifts of the adducts with cobalt(III) lie in the range −33.2 to + 49.4 ppm. This may be compared with an overall known chemical shift range in excess of 7000 ppm and implies a similarity in the molybdenum environment in all cases. For adducts with chelated cobalt(III) complexes several rather broad 95 Mo singnals are obtained with linewidths up to 260 Hz.

Isolement du Ru(OEP)(PhNO) 2 et Ru(OEP)(PhNO)py et generation in situ de Ru(OEP)(PhNO)L ou (OEP=octaethylporphyrine, py=pyridine, L=vacant, CO, H 2 O ou PPh 3 )

Isolement du Ru(OEP)(PhNO) 2 et Ru(OEP)(PhNO)py et generation in situ de Ru(OEP)(PhNO)L ou (OEP=octaethylporphyrine, py=pyridine, L=vacant, CO, H 2 O ou PPh 3 )

This presentation reviews some recent studies from these laboratories that concern activation of dihydrogen and of dioxygen by various ruthenium complexes in solution. Part 1 describes developments in the catalytic chemistry of Ru(II)/chelating ditertiary phosphine complexes, in particular their applications in asymmetric hydrogenation, while Part 2 outlines work on O 2 -oxidation of thioethers catalyzed by Ru(II) porphyrin complexes. An experimental section, giving full details on the syntheses and characterization of the complexes, including X-ray crystallographic analyses, and on the catalysis experiments, is not given in this paper; such details will appear in future publications, but are meanwhile available on request.

This presentation reviews some recent studies from these laboratories that concern activation of dihydrogen and of dioxygen by various ruthenium complexes in solution. Part 1 describes developments in the catalytic chemistry of Ru(II)/chelating ditertiary phosphine complexes, in particular their applications in asymmetric hydrogenation, while Part 2 outlines work on O 2 -oxidation of thioethers catalyzed by Ru(II) porphyrin complexes. An experimental section, giving full details on the syntheses and characterization of the complexes, including X-ray crystallographic analyses, and on the catalysis experiments, is not given in this paper; such details will appear in future publications, but are meanwhile available on request.