Chicken Liver Sulfite Oxidase Research Articles

Three-Dimensional Structural Model of Chicken Liver Sulfite Oxidase in its Activated Form

Sulfite oxidase (SO) catalyzes the conversion of sulfite to sulfate in almost all living organisms. In vertebrates, the catalytic process involves a rapid intramolecular electron transfer (IET) step between the molybdenum cofactor in the central domain and the heme in the cytochrome b5 domain. The large distance between redox centers observed in the crystal structure disagrees with the fast IET rates measured experimentally. This conflict was explained by postulating a major rearrangement of the cytochrome b5 domain toward the molybdopterin cofactor. Using steered molecular dynamics and molecular dynamics simulations, we generated a stable 3D structural model for chicken liver SO (CSO) in the activated form characterized by a short electron donor−acceptor distance consistent with the enzymes’ experimentally obtained electron transfer properties. IET rates for the active complex were estimated with the Pathway model. The good agreement between calculated and experimental IET rates supports our structural model for the active CSO.

The Structures of the C185S and C185A Mutants of Sulfite Oxidase Reveal Rearrangement of the Active Site

Sulfite oxidase (SO) catalyzes the physiologically critical conversion of sulfite to sulfate. Enzymatic activity is dependent on the presence of the metal molybdenum complexed with a pyranopterin-dithiolene cofactor termed molybdopterin. Comparison of the amino acid sequences of SOs from a variety of sources has identified a single conserved Cys residue essential for catalytic activity. The crystal structure of chicken liver sulfite oxidase indicated that this residue, Cys185 in chicken SO, coordinates the Mo atom in the active site. To improve our understanding of the role of this residue in the catalytic mechanism of sulfite oxidase, serine and alanine variants at position 185 of recombinant chicken SO were generated. Spectroscopic and kinetic studies indicate that neither variant is capable of sulfite oxidation. The crystal structure of the C185S variant was determined to 1.9 A resolution and to 2.4 A resolution in the presence of sulfite, and the C185A variant to 2.8 A resolution. The structures of the C185S and C185A variants revealed that neither the Ser or Ala side chains appeared to closely interact with the Mo atom and that a third oxo group replaced the usual cysteine sulfur ligand at the Mo center, confirming earlier extended X-ray absorption fine structure spectroscopy (EXAFS) work on the human C207S mutant. An unexpected result was that in the C185S variant, in the absence of sulfite, the active site residue Tyr322 became disordered as did the loop region flanking it. In the C185S variant crystallized in the presence of sulfite, the Tyr322 residue relocalized to the active site. The C185A variant structure also indicated the presence of a third oxygen ligand; however, Tyr322 remained in the active site. EXAFS studies of the Mo coordination environment indicate the Mo atom is in the oxidized Mo(VI) state in both the C185S and C185A variants of chicken SO and show the expected trioxodithiolene active site. Density functional theory calculations of the trioxo form of the cofactor reasonably reproducd the Mo horizontal lineO distances of the complex; however, the calculated Mo-S distances were slightly longer than either crystallographic or EXAFS measurements. Taken together, these results indicate that the active sites of the C185S and C185A variants are essentially catalytically inactive, the crystal structures of C185S and C185A variants contain a fully oxidized, trioxo form of the cofactor, and Tyr322 can undergo a conformational change that is relevant to the reaction mechanism. Additional DFT calculations demonstrated that such methods can reasonably reproduce the geometry and bond lengths of the active site.

Molecular Basis of Intramolecular Electron Transfer in Sulfite-oxidizing Enzymes Is Revealed by High Resolution Structure of a Heterodimeric Complex of the Catalytic Molybdopterin Subunit and a c-Type Cytochrome Subunit

Sulfite-oxidizing molybdoenzymes convert the highly reactive and therefore toxic sulfite to sulfate and have been identified in insects, animals, plants, and bacteria. Although the well studied enzymes from higher animals serve to detoxify sulfite that arises from the catabolism of sulfur-containing amino acids, the bacterial enzymes have a central role in converting sulfite formed during dissimilatory oxidation of reduced sulfur compounds. Here we describe the structure of the Starkeya novella sulfite dehydrogenase, a heterodimeric complex of the catalytic molybdopterin subunit and a c-type cytochrome subunit, that reveals the molecular mechanism of intramolecular electron transfer in sulfite-oxidizing enzymes. The close approach of the two redox centers in the protein complex (Mo-Fe distance 16.6 A) allows for rapid electron transfer via tunnelling or aided by the protein environment. The high resolution structure of the complex has allowed the identification of potential through-bond pathways for electron transfer including a direct link via Arg-55A and/or an aromatic-mediated pathway. A potential site of electron transfer to an external acceptor cytochrome c was also identified on the SorB subunit on the opposite side to the interaction with the catalytic SorA subunit.

Structural and Biochemical Identification of a Novel Bacterial Oxidoreductase

By using a bioinformatics screen of the Escherichia coli genome for potential molybdenum-containing enzymes, we have identified a novel oxidoreductase conserved in the majority of Gram-negative bacteria. The identified operon encodes for a proposed heterodimer, YedYZ in Escherichia coli, consisting of a soluble catalytic subunit termed YedY, which is likely anchored to the membrane by a heme-containing trans-membrane subunit termed YedZ. YedY is uniquely characterized by the presence of one molybdenum molybdopterin not conjugated by an additional nucleotide, and it represents the only molybdoenzyme isolated from E. coli characterized by the presence of this cofactor form. We have further characterized the catalytic subunit YedY in both the molybdenum- and tungsten-substituted forms by using crystallographic analysis. YedY is very distinct in overall architecture from all known bacterial reductases but does show some similarity with the catalytic domain of the eukaryotic chicken liver sulfite oxidase. However, the strictly conserved residues involved in the metal coordination sphere and in the substrate binding pocket of YedY are strikingly different from that of chicken liver sulfite oxidase, suggesting a catalytic activity more in keeping with a reductase than that of a sulfite oxidase. Preliminary kinetic analysis of YedY with a variety of substrates supports our proposal that YedY and its many orthologues may represent a new type of membrane-associated bacterial reductase.

Ground and excited state spectral comparisons of models for sulfite oxidase

Determining the contributions of thiolate and ene-1,2-dithiolate donors to the underlying electronic structure of the Mo site in sulfite oxidase (SO) has been a goal pursued by inorganic chemists for almost twenty years. The use of the hydrotris(3,5-dimethyl-1-pyrazolyl)borate ligand provided the framework for the first low-symmetry oxo-molybdenum(V) models in which the oxo donor was cis to the dithiolene moiety of benzene-1,2-dithiolate. Recent work from the Carrano and Kirk laboratories has produced a second-generation model of the SO active site based upon the (2-dimethylethanethiol)bis(3,5-dimethylpyrazolyl)methane heteroscorpionate ligand. Use of this heteroscorpionate framework provides for an oxo-molybdenum(V) complex with both a thiolate and an ene-1,2-dithiolate in the equatorial plane. In addition to providing a good structural model, the heteroscorpionate thiolate offers a constrained O–Mo–S thiolate–C torsion angle of ∼115°, closer to that of the ∼90° O–Mo–S Cys–C torsion angle observed in the chicken liver SO active site than can be obtained with thiophenolate ligands. This constrained torsion angle allows for the examination of how second-coordination sphere effects influence the electronic structure of the Mo site. Here we compare and contrast the molecular and electronic structures of these first- and second-generation models of the SO active site. Additionally, the electron paramagnetic resonance (EPR) parameters for both of these models are compared with those measured for the Mo(V) form of both low pH ( lpH) and high pH ( hpH) forms of SO in order to determine what insight these structural models can offer about the electronic structure of the active site of SO.

A voltammetric study of interdomain electron transfer within sulfite oxidase.

Protein film voltammetry of chicken liver sulfite oxidase (SO) bound at the pyrolytic graphite "edge" or modified gold electrodes shows that catalytic electron transport is controlled by the inherent electrochemical characteristics of the heme b domain and conformational changes that allow intramolecular electron transfer with the molybdenum active site. In the absence of sulfite, a single nonturnover electrochemical signal is observed at +90 mV (vs SHE) that is assigned to heme b. In the presence of sulfite, this signal transforms into a catalytic wave at similar potential. The shape and negligible pH dependence of this wave indicate that catalytic turnover is controlled by the one-electron transfers through heme b. The smaller turnover numbers obtained in this experiment (k(cat) approximately 2-4 s(-1), as compared to 100 s(-1) in solution) suggest that only a small fraction of SO is bound at the electrode in a manner that permits the conformational change necessary for fast interdomain electron transfer.

The Mo−OH proton of the low-pH form of sulfite oxidase: Comparison of the hyperfine interactions obtained from pulsed ENDOR, CW-EPR and ESEEM measurements

Pulsed electron nuclear double resonance (ENDOR) spectra have been obtained for the exchangeable Mo-OH proton of the low-pH form of native chicken liver sulfite oxidase (SO) and recombinant human SO for the first time. The spectra of the two enzymes are very similar, indicating a similar binding geometry of the hydroxyl ligand to the Mo center. The isotropic hyperfine interaction (hfi) constant for the proton of the OH ligand in both enzymes is about 26 MHz. The anisotropic components of the hfi obtained from the pulsed ENDOR spectra are about 1.6–1.8 times larger than those obtained by continuous-wave electron paramagnetic resonance and electron spin echo envelope modulation. These hfi differences are explained by a rotational disorder of the Mo-OH group. A similar rotational disorder of the coordinated exchangeable ligand has been found previously for the high-pH and phosphate-inhibited forms of SO.

Pulsed EPR studies of nonexchangeable protons near the Mo(V) center of sulfite oxidase: direct detection of the alpha-proton of the coordinated cysteinyl residue and structural implications for the active site.

Pulsed electron nuclear double resonance (ENDOR) spectra of nonexchangeable protons in the vicinity of the Mo(V) center of the high pH (hpH) and low pH (lpH) forms of native chicken liver sulfite oxidase (SO) and recombinant human SO have been obtained and analyzed for the first time. The close similarity of the spectra for the chicken and human enzymes indicates that the structures of their molybdenum centers are essentially identical. For lpH SO, the closest nonexchangeable proton is found to be approximately 2.8 A from the Mo atom. To more accurately determine the distance to this proton and facilitate its assignment, the C-band electron spin-echo envelope modulation (ESEEM) spectra of lpH SO were also analyzed. From the obtained distance and comparison with the X-ray structure, this closest nonexchangeable proton is assigned to the alpha-proton of the coordinated conserved cysteinyl residue (Cys185 in chicken, Cys207 in human). The closest Mo...H distance for the nonexchangeable protons of hpH SO is found to be approximately 3.3 A. For the cysteinyl alpha-proton, such an increase in the Mo...H distance only requires a very small change in torsional angles. This study demonstrates that details of the enzyme structural rearrangements with pH can be monitored by ENDOR spectroscopy and suggests that a similar approach may be routinely used to probe the orientation of the coordinated cysteinyl residue in mutant forms of SO that are catalytically compromised.

Effect of solution viscosity on intramolecular electron transfer in sulfite oxidase.

Our previous studies have shown that the rate constant for intramolecular electron transfer (IET) between the heme and molybdenum centers of chicken liver sulfite oxidase varies from approximately 20 to 1400 s(-1) depending upon reaction conditions [Pacheco, A., Hazzard, J. T., Tollin, G., and Enemark, J. H. (1999) J. Biol. Inorg. Chem. 4, 390-401]. These two centers are linked by a flexible polypeptide loop, suggesting that conformational changes, which alter the Mo-Fe distance, may play an important role in the observed IET rates. In this study, we have investigated IET in sulfite oxidase using laser flash photolysis as a function of solution viscosity. The solution viscosity was varied over the range of 1.0-2.0 cP by addition of either polyethylene glycol 400 or sucrose. In the presence of either viscosogen, an appreciable decrease in the IET rate constant value is observed with an increase in the solvent viscosity. The IET rate constant exhibits a linear dependence on the negative 0.7th power of the viscosity. Steady-state kinetics and EPR experiments are consistent with the interpretation that viscosity, and not other properties of the added viscosogens, is responsible for the dependence of IET rates on the solvent composition. The results are consistent with the role of conformational changes on IET in sulfite oxidase, which helps to clarify the inconsistency between the large rate constant for IET between the Mo and Fe centers and the long distance (approximately 32 A) between these two metal centers observed in the crystal structure [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garnett, R. M., Rajagopalan, K. V., Enemark, J. H., and Rees, D. C. (1997) Cell 91, 973-983].

Pulsed ELDOR spectroscopy of the Mo(V)/Fe(III) state of sulfite oxidase prepared by one-electron reduction with Ti(III) citrate.

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 electronic 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 approximately 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 reduced Mo(V)/Fe(III) form of SO by reduction with Ti(III) has enabled the interaction between these two paramagnetic metal centers, which are linked by a flexible 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 several microwave frequencies by pumping on the narrow Mo(V) signal and observing the effect on the Fe(III) primary echo at both higher and lower frequencies 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 being the dominant dipolar interaction. However, thorough analysis of the ELDOR decay curves and simulations suggests a distribution of intramolecular Mo...Fe distances, consistent with the proposal of multiple conformations in solution for the flexible loop that connects the Mo and heme domains of SO.

Direct Detection of the Proton-Containing Group Coordinated to Mo(V) in the High pH Form of Chicken Liver Sulfite Oxidase by Refocused Primary ESEEM Spectroscopy: Structural and Mechanistic Implications

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 protons and where high spectral resolution is not required. The distinctive differences in the CW and pulsed EPR spectra of the lpH and hpH forms are proposed to result from differences of the MoV−OH torsional angle and variations in the H-bonding interactions, which control the orientation of the MoV−OH proton(s) relative to the half-filled 4dxy orbital. The large isotropic hfi for the lpH form is suggested to result from an intramolecular MoV−OH···Scys hydrogen bonding interaction that places the proton of the MoV−OH group in the equatorial plane of the square pyramidal oxo-Mo(V) center of SO.

The pH dependence of intramolecular electron transfer rates in sulfite oxidase at high and low anion concentrations.

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.

ESEEM Investigations of the High pH and Low pH Forms of Chicken Liver Sulfite Oxidase

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...

Molecular Basis of Sulfite Oxidase Deficiency from the Structure of Sulfite Oxidase

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 reaction of chicken liver sulfite oxidase with dimethylsulfite

We have undertaken a steady-state and rapid kinetic study of the reaction of enzyme with sulfite and dimethylsulfite. Methylation of sulfite results in a significant increase in K m and K d for the substrate in the course of steady-state and rapid reaction kinetics, respectively, but k cat and the limiting rate constant for enzyme reduction ( k red) are essentially unchanged. This indicates that while substrate oxyanion groups are effective in stabilizing the E ox · S complex, the breakdown of this complex proceeds at the same rate even in their absence. The critical element of the substrate required for reactivity is a suitable lone-pair available to undertake nucleophilic attack on a MoO group of the active site.

Quantitation of molybdopterin oxidation product in wild-type and molybdenum cofactor deficient mutants of Chalamydomonas reinhardtii

A simple and reliable procedure of oxidation of molybdenum cofactor (MoCo) from molybdoenzymes by autoclaving samples at 120°C for 20 min yielded a single predominant fluorescent species that could be quantitatively determined by reverse phase high performance liquid chromatography. This method allowed detection and quantitation of molybdopterin in cell-free extracts of the green alga Chlamydomonas reinhardtii. The MoCo oxidation product from C. reinhardtii has the same chromatographic and spectral properties as that of milk xanthine oxidase and chicken liver sulfite oxidase. The oxidized species was also detected in molybdenum cofactor mutants of Chlamydomonas reinhardtii defective at the nit-3, nit-4, nit-5/ nit-6 and nit-7 loci, which strongly suggests that active molybdenum cofactor itself is not directly involved in the control of its own biosynthetic pathway.

A conserved cysteine in molybdenum oxotransferases.

The amino acid sequences of peptides derived from rat hepatic sulfite oxidase have been determined by a combination of amino acid analysis and Edman degradation of the purified protein. The data obtained showed the rat liver enzyme contained 3 cysteine residues which was confirmed by thiol modification studies using 4,4'-dithiodipyridine of the native enzyme. Combining these data with that previously published for chicken liver sulfite oxidase (Neame, P. J., and Barber, M. J. (1989) J. Biol. Chem. 264, 20894-20901) indicates that 2 cysteines (Cys186 and Cys430, based upon the numbering for the chicken sequence) are conserved in both chicken and rat liver enzymes with all the cysteine residues being present in the molybdenum-containing domain. Further comparison of the sequences of the molybdenum domains of rat and chicken liver sulfite oxidase with the amino acid sequences published for the molybdenum domains of a variety of assimilatory nitrate reductases suggests that only a single cysteine residue (Cys186) is conserved in all these enzymes, indicating that it may play a role in the binding of Mo-pterin to the protein.

Molybdopterin guanine dinucleotide: a modified form of molybdopterin identified in the molybdenum cofactor of dimethyl sulfoxide reductase from Rhodobacter sphaeroides forma specialis denitrificans.

The nature of molybdenum cofactor in the bacterial enzyme dimethyl sulfoxide reductase has been investigated by application of alkylation conditions that convert the molybdenum cofactor in chicken liver sulfite oxidase and milk xanthine oxidase to the stable, well-characterized derivative [di(carboxamidomethyl)]molybdopterin. The alkylated pterin obtained from dimethyl sulfoxide reductase was shown to be a modified form of alkylated molybdopterin with increased absorption in the 250-nm region of the spectrum and altered chromatographic behavior. The complex alkylated pterin was resolved into two components by treatment with nucleotide pyrophosphatase. These were identified as di(carboxamidomethyl)molybdopterin and GMP by their absorption spectra, coelution with standard compounds, and by further degradation by alkaline phosphatase to dephospho [di(carboxamidomethyl)]molybdopterin and guanosine. The GMP moiety was sensitive to periodate, identifying it as the 5' isomer. Chemical analysis of the intact alkylated pterin showed the presence of two phosphate residues per pterin. These results established that the pterin isolated from dimethyl sulfoxide reductase contains the phosphoric anhydride of molybdopterin and 5'-GMP, which is designated molybdopterin guanine dinucleotide.

Chicken liver sulfite oxidase. Kinetics of reduction by laser-photoreduced flavins and intramolecular electron transfer.

Laser flash photolysis was used to study the reaction of photoproduced 5-deazariboflavin (dRFH.), lumiflavin (LFH.), and riboflavin (RFH.) semiquinone radicals with the redox centers of purified chicken liver sulfite oxidase. Kinetic studies of the native enzyme with dRFH. yielded a second-order rate constant of 4.0 X 10(8) M-1 s-1 for direct reduction of the heme and a first-order rate constant of 310 s-1 for intramolecular electron transfer from the Mo center to the heme. The reaction with LFH. gave a second-order rate constant of 2.9 X 10(7) M-1 s-1 for heme reduction. Reoxidation of the reduced heme due to intramolecular electron transfer to the Mo center gave a first-order rate constant of 155 s-1. The direction of intramolecular electron transfer using dRFH. and LFH. was independent of the buffer used for the experiment. The different first-order rate constants observed for intramolecular electron transfer using dRFH. and LFH. are proposed to result from chemical differences at the Mo site. Flash photolysis studies with cyanide-inactivated sulfite oxidase using dRFH. and LFH. resulted in second-order reduction of the heme center with rate constants identical with those obtained with the native enzyme, whereas the first-order intramolecular electron-transfer processes seen with the native enzyme were absent. The isolated heme peptide of sulfite oxidase gave only second-order kinetics upon laser photolysis and confirmed that the first-order processes observed with the native enzyme involve the Mo site.(ABSTRACT TRUNCATED AT 250 WORDS)

Methionine-oxidized horse heart cytochromes c. I. Reaction with Chloramine-T, products, and their oxidoreduction properties

Spectroscopically, the modification of horse heart ferricytochrome c with N-chloro-4-toluolsul-fonamide (Chloramine-T, CT) occurs through a two-step process, the disruption of the methionine-80 sulfur-iron linkage and a reagent-independent change, an intramolecular rearrangement. Chromatographic purification of the preparation at a 2.5:1 reagent-to-protein ratio, pH 8.0–8.5, yields two major products, the FII and FIII CT-cytochromes c. Both products contain modification of only the methionines, 80 and 65, to sulfoxides; both are monomeric, reduced by ascorbate, and the ferrous forms are oxidized by molecular oxygen and bind carbon monoxide. The redox potentials of FII and FIII are 135 and 175±15 mV. The FIII is indistinguishable from the native protein in its binding and the electron donor property toward mammalian cytochrome c oxidase. It also binds nearly as effectively as the native protein to yeast cytochrome c peroxidase, but is a less efficient donor. It is, however, a poor electron acceptor from both mammalian cytochrome c reductase and chicken liver sulfite oxidase. FII lacks cytochrome c oxidase activity and is also a poorer substrate for the other three enzymes. Both the derivatives are consistently better electron donors than acceptors. It is concluded that the binding of cytochrome c to cytochrome c oxidase and to cytochrome c peroxidase does not require the integrity of the methionine-80 sulfur linkage and that the complexation process has a finite degree of freedom with regard to the state of the heme crevice opening. The alterations of the oxidoreduction function have been analyzed in light of both prevailing models of cytochrome c function, the two-site model (one site for oxidizing and the other for reducing enzymes) and the single-site model (the same site for the oxidizing and reducing enzymes). These observations can be accommodated by either model, given the latitude that the binding domains for the oxidizing and the reducing enzymes have finite overlapping and nonoverlapping regions.