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 Å) 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. 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 Å) 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. Sulfite oxidases (SO(s)) 1The abbreviations used are: SO, sulfite oxidase; Moco, molybdopterin cofactor; CSO, chicken liver sulfite oxidase; HSO, human liver sulfite oxidase; PSO, plant sulfite oxidase; IET, intramolecular electron transfer; r.m.s.d., root mean square deviation. 1The abbreviations used are: SO, sulfite oxidase; Moco, molybdopterin cofactor; CSO, chicken liver sulfite oxidase; HSO, human liver sulfite oxidase; PSO, plant sulfite oxidase; IET, intramolecular electron transfer; r.m.s.d., root mean square deviation. and nitrate reductases are members of a subclass of molybdopterin enzymes characterized by the presence of a MoO2 metal center. The enzymes typically contain a molybdopterin cofactor (Moco) implicated in substrate binding and oxidation and a heme cofactor for molybdenum reoxidation. Three types of sulfite-oxidizing enzymes have been characterized so far, namely the homodimeric, heme b and molybdenum-containing enzymes from higher animals and birds (1Rajagopalan K.V. Coughlan M.P. Molybdenum and Molybdenum-Containing Enzymes. Pergamon Press, Oxford1980: 243-272Google Scholar, 2Kisker C. Schindelin H. Pacheco A. Wehbi W.A. Garrett R.M. Rajagopalan K.V. Enemark J.H. Rees D.C. Cell. 1997; 91: 973-983Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar), the homodimeric, molybdenum-containing enzymes from plants (3Eilers T. Schwarz G. Brinkmann H. Witt C. Richter T. Nieder J. Koch B. Hille R. Haensch R. Mendel R.R. J. Biol. Chem. 2001; 276: 46989-46994Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) and a heterodimeric, heme c and molybdenum-containing bacterial enzyme (4Kappler U. Bennett B. Rethmeier J. Schwarz G. Deutzmann R. McEwan A.G. Dahl C. J. Biol. Chem. 2000; 275: 13202-13212Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) that cannot transfer electrons to molecular oxygen and is therefore classified as a sulfite dehydrogenase. Of these enzymes, the sulfite oxidases from higher animals and birds have been studied in great detail, as in humans a deficiency in SO activity is linked to severe developmental retardation and early death of the affected individuals (2Kisker C. Schindelin H. Pacheco A. Wehbi W.A. Garrett R.M. Rajagopalan K.V. Enemark J.H. Rees D.C. Cell. 1997; 91: 973-983Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 5Mize C. Johnson J.L. Rajagopalan K.V. J. Inherit. Metab. Dis. 1995; 18: 283-290Crossref PubMed Scopus (27) Google Scholar). The crystal structures of two homodimeric eukaryotic sulfite oxidases, CSO from chicken liver (2Kisker C. Schindelin H. Pacheco A. Wehbi W.A. Garrett R.M. Rajagopalan K.V. Enemark J.H. Rees D.C. Cell. 1997; 91: 973-983Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar) and PSO from Arabidopsis thaliana (6Schrader N. Fischer K. Theis K. Mendel R.R. Schwarz G. Kisker C. Structure. 2003; 11: 1251-1263Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), have been determined and have provided structural details of the molybdenum center and substrate binding pocket. During catalysis two electrons are transferred from the substrate, sulfite, to the fully oxidized Mo(VI) form of the active site, which is then reduced to the Mo(IV) state. The two electrons are then transferred sequentially from the molybdenum to an electron acceptor (PSO) or to the heme cofactor, which can be present either as a domain (e.g. CSO, human sulfite oxidase (HSO)) or subunit (e.g. bacterial sulfite dehydrogenase) of the enzyme. Using the structural information and the results from site-directed mutagenesis studies on HSO (7Feng C.J. Wilson H.L. Hurley J.K. Hazzard J.T. Tollin G. Rajagopalan K.V. Enemark J.H. J. Biol. Chem. 2003; 278: 2913-2920Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 8Feng C.J. Wilson H.L. Hurley J.K. Hazzard J.T. Tollin G. Rajagopalan K.V. Enemark J.H. Biochemistry. 2003; 42: 12235-12242Crossref PubMed Scopus (48) Google Scholar, 9Wilson H.L. Rajagopalan K.V. J. Biol. Chem. 2004; 279: 15105-15113Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) it has been possible to assign functions, especially in substrate binding, to various residues that are conserved in the vicinity of the active site. Intramolecular electron transfer (IET) between Moco and the heme is fundamental to the function of the mammalian and bacterial enzymes; however, the electron transfer pathway between the molybdenum and the heme cofactor has not yet been determined. One reason for this is the way in which the heme domain is attached to the rest of the protein in CSO and HSO, namely as a separate domain that is connected to the Moco/dimerization domains of the protein by a flexible linker that allows for extensive changes in its position relative to the other two domains of the protein. In fact, in the published CSO structure the position of the heme domain is removed from the active site (2Kisker C. Schindelin H. Pacheco A. Wehbi W.A. Garrett R.M. Rajagopalan K.V. Enemark J.H. Rees D.C. Cell. 1997; 91: 973-983Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 10Schindelin H. Kisker C. Rajagopalan K.V. Adv. Protein Chem. 2001; 58: 47-94Crossref PubMed Scopus (66) Google Scholar) with a distance of 32 Å between the two metal centers. This distance cannot be expected to allow for efficient electron transfer, and this enzyme structure has been presumed to be in a “redox-inactive” state. The viscosity of the medium has a marked effect on IET in CSO, which suggests that domain movement is essential for catalysis in this protein (11Feng C.J. Kedia R.V. Hazzard J.T. Hurley J.K. Tollin G. Enemark J.H. Biochemistry. 2002; 41: 5816-5821Crossref PubMed Scopus (107) Google Scholar). At present, it is unknown how the heme domain of the vertebrate enzymes is attached to the Moco domain during transfer of electrons or how this domain interacts with its external electron acceptor, cytochrome c. Here we present a description of the crystal structures of the reduced state of the heterodimeric sulfite dehydrogenase (SorAB) from the soil bacterium Starkeya novella (4Kappler U. Bennett B. Rethmeier J. Schwarz G. Deutzmann R. McEwan A.G. Dahl C. J. Biol. Chem. 2000; 275: 13202-13212Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). This structure is the first for a multisubunit sulfite-oxidizing enzyme and allows direct insights into the electron transfer pathway between the molybdenum and heme centers and the nature of the subunit interface in such enzymes. Purification and Crystallization—Recombinant sulfite dehydrogenase was purified and crystallized as described previously (12Kappler U. Bailey S. Acta Crystallogr. Sect. D. 2004; 60: 2070-2072Crossref PubMed Scopus (10) Google Scholar). To generate fully oxidized protein samples, purified protein was mixed with excess cytochrome c (horse heart, C-7752, Sigma), incubated on ice for 1 h and then subjected to size-exclusion chromatography (Superdex 75 6/60 prep grade, GE Healthcare) to remove the horse heart cytochrome. Data Collection—Data were collected on beamlines at the Advanced Light Source, Berkeley and at the SRS, Daresbury Laboratory. Although three-wavelength multiwavelength anomalous diffraction data were collected for the sodium bromide-soaked crystal, only one peak wavelength was used in the structure determination. All data were processed using the program and on Protein Scholar) and and using the Acta Crystallogr. Sect. D. PubMed Scopus Google Scholar). Data and are in and was reduced by the presence of sodium sulfite in the and were collected on the high side of the was oxidized with cytochrome c to were reduced by the of 1 sodium sulfite 1 to in to the resolution in to the resolution in to the resolution in to the resolution and are the and structure was with of the data that been from from of Protein was reduced by the presence of sodium sulfite in the and Data were collected on the high side of the in to the resolution and are the and structure was with of the data that been from Protein was oxidized with cytochrome c to were reduced by the of 1 sodium sulfite 1 to in a crystal structure has been determined by a of molecular and The for molecular was from the structure of CSO The molecular determined using the program Acta Crystallogr. Sect. D. 2001; PubMed Scopus Google Scholar, A. A. J. Crystallogr. 1997; Scopus Google Scholar), was using the Moco domain of CSO by of protein side to the between the two The position of the domain was determined using the program K. Acta Sect. D. 2001; PubMed Scopus Google Scholar). in the be determined from the of in and anomalous using the molecular The position of the cytochrome was also determined from anomalous using data collected on the high side of the The molecular and were used with the Å resolution data as the for using A. Biol. PubMed Scopus Google Scholar) a The residues and the were using the program Acta Crystallogr. Sect. A. PubMed Scopus Google Scholar). was using the program Acta Sect. D. 1997; PubMed Scopus Google Scholar), and are in The data in were used for structure and The of Moco is well however, the molybdenum is not fully in this crystal, and with an of a The of residues of the SorA subunit, residues of the SorB subunit, one Moco, one c-type a sulfate and Three were identified at and of the SorA and a is present between and close to the protein The structures have with of the residues in the and allowed and residues in of the as by J. Crystallogr. Google Scholar). The structures are well as by the and by the electron of the protein structure were with the program and were to structures of in the oxidized and reduced and of either data not or sulfite to the protein during to to a active site with of structures of enzyme and reduced with sulfite for these and structures with fully molybdenum and structure have been in the Protein Data and of structure of the sulfite dehydrogenase has been determined using a of molecular and The structures of and enzyme and structures with the reduced state of Moco, of by the during data of the structure described in this are from the the data in is a complex of two a Moco binding subunit, and a cytochrome subunit (4Kappler U. Bennett B. Rethmeier J. Schwarz G. Deutzmann R. McEwan A.G. Dahl C. J. Biol. Chem. 2000; 275: 13202-13212Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). the catalytic subunit, two domains that the domains of CSO and PSO (2Kisker C. Schindelin H. Pacheco A. Wehbi W.A. Garrett R.M. Rajagopalan K.V. Enemark J.H. Rees D.C. Cell. 1997; 91: 973-983Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, N. Fischer K. Theis K. Mendel R.R. Schwarz G. Kisker C. Structure. 2003; 11: 1251-1263Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). in domain 1 has a mixed and is for binding Moco domain has an structure with the as an one of domain that from the into the Moco domain and to the of the substrate binding is for of the interface in the homodimeric eukaryotic sulfite oxidases has function in and structure of of mammalian plant and bacterial sulfite-oxidizing enzymes, of the nitrate reductases and the sulfur also to the sulfite oxidase protein site residues are residues to the molybdenum cofactor are in with the conserved active site is in a in are in and residues in that between the CSO, and PSO are with a The SorB subunit has a c-type cytochrome A.G. T. C. J. Biol. 1995; PubMed Scopus Google Scholar) and is in with the of the which from the of the subunit and the Moco SorB the heme binding of a cytochrome namely the close to the and a c-type heme with The heme is attached by to the two conserved residues and A of the protein data that SorB is to the of A of dehydrogenase from S. K. K. T. S. T. K. K. U. S. A. 2001; PubMed Scopus Google Scholar), which This is of a cytochrome domain and can be with an of One of these is the of between 1 and and and which are present in the of heme bacterial c-type and the heme cofactor from of the heme of SorB are to which are present at the of the heme in the cytochrome also be to however, in the provided by the Moco domain interacts with the cytochrome at this of the the of sulfite dehydrogenase SorB has and structural with other c-type between SorA and complex can be purified from S. novella and is and a subunit interaction that is and of high This is the first of a sulfite-oxidizing protein the cytochrome is present as a separate subunit and in a position to IET, and therefore the nature of the subunit interface in the complex is of The cytochrome interacts only with the Moco domain of an interface of two One between the of the cytochrome and the is provided by the of the cytochrome which Å the of SorA and and is of the subunit of the heterodimeric nitrate from J. B. B. D. Biol. 2003; PubMed Scopus Google Scholar). The on complex is with by 1 and by extensive are with a heterodimeric complex S. J. Biol. Scopus Google Scholar). A of direct including two and are formed at the subunit a with an of in a of 75 C. J. J. Biol. PubMed Scopus Google Scholar) One of the between and is at the between the and of the and residues are conserved in The interface between the of the cytochrome and SorA of an to the substrate binding site of the cytochrome not close with SorA in this and substrate are to the between the two on one side of the interface The heme into this the heme to and of the between Arg-55A and the heme The two to the of the at the interface of the SorA and SorB of the of CSO, and domains of subunit SorA have the as the Moco and domains of CSO and PSO, and can be on these domains with an of and Å for CSO and PSO, the Moco and domains are the is a in the relative of these domains that has not been in the CSO or PSO structures of Moco and domains of on CSO and residues Moco domain residues domain residues Moco and in a Although the in the structural of sulfite-oxidizing enzymes are in and the position of the 1 and residues and in and are not in A residues is also in and in this and the residues form with the cytochrome subunit. Of are the structural for the residues which the in CSO and PSO and at the interface of these enzymes. In the heterodimeric this an between and and the that the of a interface in The arises as to the function of this domain and the in other so as it is not for catalytic function in which is a highly active and sulfite dehydrogenase. A is on CSO is the position of the cytochrome subunit relative to the Moco domain and the active site Although in the CSO structure the heme b domain is 32 Å from the molybdenum site, in the cytochrome subunit interacts with the Moco domain to the active site and one side of the to the substrate binding site. The relative position of the SorA and SorB allows a close approach of the two The distance is 16.6 and the approach between the molybdenum and the of the heme cofactor is The distance and the presence of between the two that are in the for catalysis PubMed Scopus Google Scholar). A of the of CSO and that the have including heme and In fact, the position of SorB is to a position for the CSO heme b domain A. Hazzard J.T. Tollin G. Enemark J. Biol. Chem. PubMed Scopus Google Scholar). to the cytochrome domain of CSO into that position by of the CSO heme on the heme only on these results it that the cytochrome of CSO close to the CSO active site and that the heme is in a interaction to that for has a molybdopterin cofactor that is and in the Moco The molybdopterin is not to a which is present in bacterial molybdoenzymes to the oxidase A.G. U. D. in and Scholar, Theis K. S. R. Rajagopalan K.V. Kisker C. Structure. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). are a of between the and of the cofactor and the protein. the of the of in SorA for of CSO, the residues with side in Moco binding are conserved with other sulfite oxidases and plant nitrate reductases (2Kisker C. Schindelin H. Pacheco A. Wehbi W.A. Garrett R.M. Rajagopalan K.V. Enemark J.H. Rees D.C. Cell. 1997; 91: 973-983Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, N. Fischer K. Theis K. Mendel R.R. Schwarz G. Kisker C. Structure. 2003; 11: 1251-1263Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The molybdenum has in an square two from the an and a which is a or on the distance to the molybdenum A and In oxidized of the sulfite oxidase contain a center Enemark J.H. Biochemistry. PubMed Scopus Google Scholar), and the presence of a or is in with the expected reduced state of the of the presence of 1 The is Å from the molybdenum and is to the residues and N. The or is Å from the molybdenum and with Arg-55A and two or in the substrate binding medium we have and 1 sulfite is to the however, is for either in the substrate binding site of the which is sulfate is an of The substrate binding site was therefore identified by with the CSO structure sulfate was close to the molybdenum of the and CSO protein and conserved residues an of the CSO substrate reveals a close with an of Å for A of residues have side Å of the sulfate in and CSO, and of these are and Three of these residues and are conserved sulfite-oxidizing enzymes and nitrate reductases and form direct with the sulfate in and Arg-55A with the or to the molybdenum center. mutagenesis of the of Arg-55A and side in HSO has a effect on a role in substrate binding C.J. Wilson H.L. Hurley J.K. Hazzard J.T. Tollin G. Rajagopalan K.V. Enemark J.H. Biochemistry. 2003; 42: 12235-12242Crossref PubMed Scopus (48) Google Scholar, C. Wilson H.L. Hurley J.K. Hazzard J.T. Tollin G. Rajagopalan K.V. Enemark J.H. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus (32) Google Scholar). is conserved in with by its with Moco a role in substrate of SorA is a for the in this In SorA and CSO, the side of this a with Arg-55A and residues that the substrate binding site and that are conserved in eukaryotic sulfite-oxidizing enzymes are in The of these residues a side in CSO with a side in in a binding site for SorA A and for the mechanism of is the of and by in The of this in eukaryotic has been to be for substrate this in the of the active site for is of fundamental for studies of these enzymes and of CSO also interacts with the substrate and is by in direct with the substrate are in SorA to in sulfite dehydrogenase is Å from the position of the CSO it form an interaction to the is conserved in eukaryotic sulfite oxidases and nitrate is conserved in eukaryotic sulfite oxidases is by a in nitrate residues the substrate binding site of SorA are and CSO and The substrate binding site of SorA a of close to the sulfate position of CSO and are on substrate Three or other the by the side of and of CSO, and two of these are to be distance of the are present in the to the substrate binding site, a of well between the two of Intramolecular the interaction and the molecular of the redox centers it is possible to the of the intramolecular electron transfer The distance between the of the two in the the molybdenum of the molybdopterin and of the heme is A of redox with structures has that electrons can to Å between redox centers the protein medium and that redox center is to allow tunnelling of electrons at the substrate redox PubMed Scopus Google Scholar). suggests that pathways between the redox can be identified the protein structure 2003; PubMed Scopus Google Scholar). between the heme and SorA are by and the cofactor distance are in this Arg-55A Arg-55A and is also in a position to to the is to be and a conserved to the The of the of Arg-55A to the and molybdenum are and and in to the Arg-55A with the substrate and with the of the molybdenum center. is this that is in sulfite dehydrogenase catalysis by the of an by the substrate the interaction between the redox centers and a possible pathway. pathway for between molybdenum and a of and which the between the two redox centers with of Å The of the two residues at the of this and the cofactor and of the heme and studies in this electron transfer between the molybdenum and heme domains in bacterial and vertebrate sulfite-oxidizing enzymes. sulfite to with an external electron acceptor which has been identified as a cytochrome for of the SorB heme and the molybdenum center (4Kappler U. Bennett B. Rethmeier J. Schwarz G. Deutzmann R. McEwan A.G. Dahl C. J. Biol. Chem. 2000; 275: 13202-13212Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, T. T. K. Scholar). possible binding for an external acceptor, the SorB heme is to the can be One of these is in the substrate binding however, is to allow a close approach of the cytochrome The is on the opposite side of from the molybdenum active site is and that the this is for with the cytochrome that this position is the cytochrome c interaction site. of catalytic activity and IET in CSO (1Rajagopalan K.V. Coughlan M.P. Molybdenum and Molybdenum-Containing Enzymes. Pergamon Press, Oxford1980: 243-272Google Scholar, C.J. Kedia R.V. Hazzard J.T. Hurley J.K. Tollin G. Enemark J.H. Biochemistry. 2002; 41: 5816-5821Crossref PubMed Scopus (107) Google Scholar), in sulfate not to IET in as by C.J. U. Tollin G. Enemark J. Chem. 2003; PubMed Scopus Google Scholar) has been C.J. Wilson H.L. Hurley J.K. Hazzard J.T. Tollin G. Rajagopalan K.V. Enemark J.H. Biochemistry. 2003; 42: 12235-12242Crossref PubMed Scopus (48) Google Scholar, C.J. Kedia R.V. Hazzard J.T. Hurley J.K. Tollin G. Enemark J.H. Biochemistry. 2002; 41: 5816-5821Crossref PubMed Scopus (107) Google Scholar, C. Schindelin H. Rees D.C. 1997; PubMed Scopus Google Scholar) that the effect of sulfate on CSO IET can be by sulfate in the substrate binding close to the molybdenum center the potential in this vicinity and for the heme The published CSO two sulfate one is close to the molybdenum and the substrate binding site, and the is Å from at the of the to the substrate binding in a by two in the of the and CSO this sulfate is Å from the position of the heme of SorB it is to with the of the The binding of sulfate at this position is not possible in of the of the complex and the of the heme it is possible that binding of sulfate at the of the substrate binding to the in potential and to sulfate of IET in structure of a complex of the catalytic SorA subunit with its electron transfer the c-type cytochrome SorB subunit is and molecular insights into the mechanism of IET in sulfite-oxidizing enzymes. Although the subunit structure of the heterodimeric with the homodimeric structure of the eukaryotic has that the enzymes catalytic (4Kappler U. Bennett B. Rethmeier J. Schwarz G. Deutzmann R. McEwan A.G. Dahl C. J. Biol. Chem. 2000; 275: 13202-13212Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). the structural data it is that Arg-55A has a role in of oxidation of sulfite and transfer of electrons from Mo(IV) to the heme of the in HSO previously identified it as in substrate binding and also electron transfer in such a structure for the first how Arg-55A is in electron its direct interaction with of the heme Arg-55A to the subunit interface and the redox into an position for electron transfer to the interaction of Arg-55A with the heme and the active of the molybdenum center that it is a for of catalysis in and sulfite Arg-55A is also to a role in the pathway between redox centers IET a distance of 16.6 with the we that the eukaryotic form a A. G. S. S. J. and C. for to