Sulfite oxidase active site structure

Figure 17.2 The structure of the pterin cofactor (1) which is common to most molybdenum- and tungsten-containing enzymes and schematic active site structures for members of the xanthine oxidase (2,3), sulfite oxidase (4) and DMSO reductase (5-7) enzyme families. (From Enemark et al., 2004. Copyright (2004) American Chemical Society.)...

Figure 16. Consensus oxidized active-site structures of the xanthine oxidase (XO), sulfite oxidase (SO), and DMSO reductase (DMSOR), and aldehyde oxidoreductase (AOR) families of mononuclear molybdenum and tungsten enzymes and the structure of the common ppd cofactor (41, 42). The question mark in the AOR structure indicates uncertainty in the presence of a coordinated water molecule.

Molybdenum-containing enzymes can be divided into three families, the xanthine oxidase (XO), sulfite oxidase (SO), and the DMSO reductase (DMR) families. They each have a characteristic active site structure (Figure 17.2(a)) and catalyse a particular type of reaction (see below). Whereas in eukaryotes, the pterin side chain has a terminal phosphate group, in prokaryotes, the cofactor (R in Figure 17.2(b)) it is often a dinucleotide. 

Figure 16 The biosynthesis of Moco and bis-MGD. Shown is a scheme of the biosynthetic pathway for Moco biosynthesis in bacteria and eukaryotes. The proteins involved in the reactions are colored in red for bacterial proteins and blue for human proteins. In bacteria, Moco (54) can be further modified by the attachment of, for example, GMP, forming MGD, and two equivalents of MGDare bound to molybdenum, forming the so-called bis-MGD cofactor (56). Further, Moco can be modified by the replacement of one 0x0 ligand by a sulfido ligand, forming the monooxo Moco (55). The three molybdenum containing enzyme families are divided into the xanthine oxidase, sulfite oxidase, and DMSO reductase families according to their active site structures.

Figure 6.1 Schematic active site structures of DMSO reductase (left), sulfite oxidase (middle) and xanthine oxidase (right) families, and the structure of pyranopterindithiolate (MPT).

The sulfite oxidase enzymes are widespread in Nature, and are found in plants, bacteria (the sulfite dehydrogenases) and in birds and mammals. In addition, this family also includes the assimilatory plant nitrate reductases, which have essentially similar molybdenum coordination and differ structurally in lacking an active site arginine that is present in sulfite oxidase, and in showing somewhat different active site structures on turnover. We will focus here on the animal sulfite oxidase enzymes, of which chicken and human are the best studied. In animals the enzyme is responsible for the physiologically essential oxidation of sulfite to sulfate. It is a dimer of 52 kDa subunits and resides in the mitochondrial inner-membrane space. Each monomer contains Mo associated with one molybdopterin, plus a cytochrome heme. The enzymes catalyze the following reaction, which occurs at the Mo site which is reduced from Mo(vi) to Mo(iv) in the process ... 

Figure 4.18 EXAFS Fourier transforms of the sulfite oxidase iodide complex. Transforms are Mo-S phase-corrected, with the solid line showing experimental data and the broken line the best fit. The weak EXAFS from the distant iodide can be seen as the small transform peak at 5 A. The DFT-computed active site structure, calculated using constraints based on the crystal structure, is shown in the inset with a computed total electron density iso-surface with a value of 0.02 electrons per cubic a.u.

The photoelectron spectra of model complexes for these molybdenum enzyme active sites have been investigated to gain a better understanding of their basic electronic structure and the role of the ene-dithiolate hgand. For example, the metal coordination of model complexes such as Tp MoO(tdt) are similar to the molybdenum center of sulfite oxidase, which possesses the basic structural core of a terminal oxo group cis to a 1,2-dithiolate. 

Mo(Tp )(E)(bdt)j, [Mo(Tp )(E)(tdt)], [Mo(Tp )(E)(bdtCl2)]82 83 (E = O, NO), and [MoO(qdt)(Tp )]84 have been investigated as models for various pyranopterin Mo enzyme active sites, including sulfite oxidase. Solution redox potentials and heterogeneous electron transfer rate constants for these species have been also reported.85 The interactions between the sulfur tt-orbitals of arene dithiolates and high-valent Mo in [MoO(Tp )(bdt)] have been investigated by gas-phase photoelectron spectroscopy and DFT methods in order to understand the properties of the active site of pyranopterin Mo-W enzymes.86 Temperature-dependent measurements of potential and electron-transfer rate constants are also reported for electrochemical reduction of a series of [MoO(Tp )(X,Y)] complexes.87 The molecular and electronic structures of the SO active site [MoO(Tp )(bdt)] have been also reported.88... 

Figure 8 Structure of the oxidized form of the active site of chicken liver sulfite oxidase. ...

The initial contribution to this volume provides a detailed overview of how spectroscopy and computations have been used in concert to probe the canonical members of each pyranopterin Mo enzyme family, as well as the pyranopterin dithiolene ligand itself. The discussion focuses on how a combination of enzyme geometric structure, spectroscopy and biochemical data have been used to arrive at an understanding of electronic structure contributions to reactivity in all of the major pyranopterin Mo enzyme families. A unique aspect of this discussion is that spectroscopic studies on relevant small molecule model compounds have been melded with analogous studies on the enzyme systems to arrive at a sophisticated description of active site electronic structure. As the field moves forward, it will become increasingly important to understand the structure, function and reaction mechanisms for the numerous non-canonical [ie. beyond sulfite oxidase, xanthine oxidase, DMSO reductase) pyranopterin Mo enzymes. 

The active site geometries (Figure 2.14) of the sulfite oxidizing enzymes are quite similar and examples of high-resolution X-ray crystal structures can be found for the S. novella sulfite dehydrogenase (bacterial)/ A. thaliana sulfite oxidase (plant)/ bacterial YedY/ and vertebrate sulfite oxidase. ... 

In summary, XAS has been essential in shaping our current knowledge of the sulfite oxidase family, in part because of the availability of structural information on the oxidized enzyme, whereas the crystal structures are of photoreduced enzyme, and only the Mo(v) oxidation state can be probed by EPR and derivative methods such as ESEEM. As experimental capabilities for XAS improve, we anticipate that high-resolution XAS will play an increasingly important role in our understanding of these complex systems, together with a holistic approach to structure (Section 4.3.4) in which XAS plays a part in defining the most accurate picture possible of the active site. 

Enzymes from the DMSO reductase family are distinct in several respects from those found in the xanthine oxidase and sulfite oxidase families. Members of the DMSO reductase family are only found in prokaryotes. They are also perhaps the most difficult members of the Mo and W enzyme superfamily to classify as there is so much structural diversity both at the active site and... 

Since mitochondrial cytochrome c was available commercially (horse heart muscle being the most common source) and could readily be purified to a high level, it formed the basic subject for most of the pioneering studies. Many ideas concerning the electrochemical mechanism, in particular, the mode of interaction with the electrode, have developed around the considerable wealth of information that is available [14, 18] on the structure and properties of the protein molecule. The extent to which the metal centre is buried is illustrated well in Fig. 1 which shows the 3D structure [19] of yeast (iso-1) cytochrome c and a view of the exposed active site. The major function of cytochrome c is as electron donor to cytochrome c oxidase (Complex IV), the membrane-bound enzyme that is the terminus of the aerobic respiratory chain and a site for proton translocation. Another physiological oxidant of cytochrome c (in yeasts) is cytochrome c peroxidase, a soluble enzyme whose crystal structure is known (see Sect. 7). The most important reduc-tant of cytochrome c is the cytochrome Cj component of the membrane-bound hcj complex (Complex III), but others (see Sect. 6, Scheme 5) include cytochrome b, sulfite oxidase, and flavocytochrome (lactate dehydrogenase, found in yeasts). 

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