Electron transfer in sulfite oxidase: Effects of pH and anions on transient kinetics

Sullivan, Eric P.; Hazzard, James T.; Tollin, Gordon; Enemark, John H.

doi:10.1021/bi00097a026

Cited by 54 publications

(86 citation statements)

References 38 publications

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“…3). These spectra confirm that the transient absorbance changes observed at 555 nm are directly related to reduction and reoxidation of the b-type heme prosthetic group (11). No detectable spectral contribution from the molybdenum cofactor was observed.…”

Section: Photochemical Reduction Of Human So By Deazariboflavin Semiqsupporting

confidence: 77%

Role of Conserved Tyrosine 343 in Intramolecular Electron Transfer in Human Sulfite Oxidase

Feng¹,

Wilson²,

Hurley³

et al. 2003

Journal of Biological Chemistry

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Tyrosine 343 in human sulfite oxidase (SO) is conserved in all SOs sequenced to date. Intramolecular electron transfer (IET) rates between reduced heme (Fe II ) and oxidized molybdenum (Mo VI ) in the recombinant wild-type and Y343F human SO were measured for the first time by flash photolysis. The IET rate in wild-type human SO at pH 7.4 is about 37% of that in chicken SO with a similar decrease in k cat . Steady-state kinetic analysis of the Y343F mutant showed an increase in K m sulfite and a decrease in k cat resulting in a 23-fold attenuation in the specificity constant k cat /K m sulfite at the optimum pH value of 8.25. This indicates that Tyr-343 is involved in the binding of the substrate and catalysis within the molybdenum active site. Furthermore, the IET rate constant in the mutant at pH 6.0 is only about one-tenth that of the wild-type enzyme, suggesting that the OH group of Tyr-343 is vital for efficient IET in SO. The pH dependences of IET rate constants in the wild-type and mutant SO are consistent with the previously proposed coupled electron-proton transfer mechanism.In vertebrates the molybdenum cofactor-containing enzyme sulfite oxidase (SO, 1 EC 1.8.3.1) catalyzes the oxidation of sulfite to sulfate with the reduction of two equivalents of ferricytochrome c (Equation 1) (1, 2), according to the following equation.This is the terminal reaction in the oxidative degradation of sulfur-containing compounds and is physiologically essential. The absence of SO activity in humans is characterized by dislocation of ocular lenses, mental retardation, and in severe cases, attenuated growth of the brain and early death. These severe neurological symptoms result from the inability to properly produce the molybdenum cofactor and also from point mutations in the SO protein itself (3). The x-ray crystal structure of the dimeric chicken liver SO has recently been reported (4). Each subunit consists of an N-terminal b 5 -type cytochrome domain and two larger segments that constitute the central molybdenum binding and C-terminal interface domains. The molybdenum center in SO has square pyramidal coordination geometry. Solvent access to the deeply buried active site is via a channel that opens onto the equatorial MoϪO group. As expected for binding an anionic substrate, the sulfite-binding pocket is very positively charged and consists of three arginines (Arg-138, Arg-190, and Arg-450), Trp-204, and Tyr-322. Tyr-322 is also in close proximity to the molybdenum coordination sphere (Fig. 1A).In the generally accepted catalytic cycle (Fig. 2 , followed by reduction of a second equivalent of cyt c ox , returns the enzyme to the fully oxidized resting state. We have previously shown that exogenous flavin radicals generated in situ with a laser pulse will rapidly reduce the heme center of SO by one electron (Fig. 2) and have used this unique technique to investigate IET between the molybdenum and iron centers as a function of pH values (10Ϫ12). Note that this process corresponds to the second IET step noted above. Comb...

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Section: Photochemical Reduction Of Human So By Deazariboflavin Semiqsupporting

confidence: 77%

Role of Conserved Tyrosine 343 in Intramolecular Electron Transfer in Human Sulfite Oxidase

Feng¹,

Wilson²,

Hurley³

et al. 2003

Journal of Biological Chemistry

Self Cite

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“…Additionally, the absence of a histeresis of the titration curve might designate that it is the heme domain that is in the direct ET contact with the electrode surface; that is, no other redox centre acts as a mediator between the heme and the electrode. However, considering the shown possibility of fast internal ET between the Mo and heme active sites [6][7][8][9] and solution electrochemistry of SOx in the studied system, certain probability for the electrode-heme ET mediated by Mo centre, also exists. The last can be possible if the redox potentials of heme and the Mo-cofactor overlap.…”

Section: Resultsmentioning

confidence: 97%

“…The absence of the catalysis was attributed to the inhibition effect of bivalent anions being components of the buffer solution, i.e., phosphate anions. Previously, phosphate and sulphate anions have been shown to reduce the catalytic activity of SOx towards sulphite oxidation through competitive binding in the Mo active site [7,9,11,16]. This binding was also shown to affect internal ET from Mo-cofactor to heme active site by providing SOx conformation with an increased ET distance.…”

Section: Resultsmentioning

confidence: 98%

Spectroelectrochemical study of heme- and molybdopterin cofactor-containing chicken liver sulphite oxidase

Ferapontova

Christenson

Hellmark

et al. 2004

Bioelectrochemistry

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“…This is not surprising considering the highly positively charged active site to attract sulfite molecules. It also explains the inhibitory effect of small anions such as chloride on sulfite oxidase (27,29).…”

Section: Discussionmentioning

confidence: 92%

Structural Insights into Sulfite Oxidase Deficiency

Karakaş

Wilson

Graf

et al. 2005

Journal of Biological Chemistry

149

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Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.Sulfite oxidase (SO), 3 an enzyme containing the molybdenum cofactor (Moco), catalyzes the oxidation of sulfite to sulfate, the final step in the degradation of sulfur-containing amino acids. It resides in the intermembrane space of mitochondria, where it exists as a homodimer. The crystal structure of chicken sulfite oxidase (CLSO), purified from chicken liver, showed that each subunit contains three domains. A small heme-containing N-terminal cytochrome b 5 domain (residues 3-84) is connected to the rest of the protein via a flexible 10-residue-long loop region. The central domain (residues 92-323) contains the active site of the enzyme. Finally, the C-terminal dimerization domain (residues 324 -466) displays the same topology as the C2 subtype of the immunoglobulin superfamily (1).Upon oxidation of sulfite, Mo(VI) is reduced to Mo(IV) by two electrons. The electrons are subsequently transferred to the heme Fe(III) in the cytochrome b 5 domain in a two-step reaction, which is followed by transfer of the electrons from Fe(II) to cytochrome c (2). The distance between the two metals, molybdenum and iron, in the crystal structure of chicken liver sulfite oxidase (CLSO) is 32 Å, which is much longer than expected for the electron transfer rate observed (1). Two mechanisms were suggested to explain these results; a very efficient electron transfer through main ...

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Electron transfer in sulfite oxidase: Effects of pH and anions on transient kinetics

Cited by 54 publications

References 38 publications

Role of Conserved Tyrosine 343 in Intramolecular Electron Transfer in Human Sulfite Oxidase

Role of Conserved Tyrosine 343 in Intramolecular Electron Transfer in Human Sulfite Oxidase

Spectroelectrochemical study of heme- and molybdopterin cofactor-containing chicken liver sulphite oxidase

Structural Insights into Sulfite Oxidase Deficiency

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