Structure of the water-soluble fragment of the Rieske Iron Sulfur Protein

General Information based on Rasmol:  Spacefill display of the Rieske

Iron Sulfur Protein (ISP):  CPK colour scheme,red = oxygen, blue = Nitrogen, grey = Carbon, sulfur = yellow
The Rieske ISP is a 1169 atom, 129 residue protein fragment of MW 14402 Daltons which is involved in electron transfer.

Below is a ribbon display (Rasmol) of Rieske ISP with the Iron-Sulfur cluster  shown in lower left corner in cyan (left image).   This is the active site (cytochrome b(6)f domain) of the protein in which the electron transfer takes place which is referred to as the [2Fe-2S] cluster.  This protein has a novel fold with 3 b-sheets.  The group colour scheme is utilized to observe the residues by their position in the chain.  The red indicates the beginning of the chain, or the N-terminus while the green indicates the C-terminus.  The right image is a rotated view of the Rieske ISP which shows the ant-parallel b-strands and flanked by a single a-helix.

Resolution:  1.5Å
R-factor:  0.187 where R =å(II F_hkl(obs) I – I F_hkl(calc) II)  (Usually ~0.2)  This equation compares calculated with measured structure factors.  The (F_hkl) values are
                                                åI F_hkl(obs) I
intensities at a single spot on the diffraction pattern from X-ray scattering off of the crystal.
Space Group:  P 1 21 1 out of 65 possible for proteins.
Unit cell dimensions (Å):a32.10   b53.00  c38.00 angles: a 90.00  b 100.03  g 90.00
The MAD (multiwavelength anomalous dispersion) Phasing technique was used to obtain a crystal structure and refined against the diffraction pattern to a resolution of 1.5Å.  (See electron density map below which was used to aid in atomic modeling.)  For further information on MAD phasing techniques and calculations go to the following website:  http://www.bmsc.washington.edu/scatter/MAD_1.html
Completenes of Data:  75.6%
 
 

                        1 VLAMSKIEIK LSDIPEGKNM AFKWRGKPLF VRHRTKKEID QEAAVEVSQL
                                      SSEEEE GGGTTTT EE EEEETTEEEE EEE  HHHHH HHHTTTGGG

                      51 RDPQHDLERV KKPEWVILIG VCTHLGCVPI ANAGDFGGYY CPCHGSHYDA
                           SS   GGGT  SSTTEEEEE    TTTS   E ETTTTTSSEE ETTTTEEE T

                    101 SGRIRKGPAP LNLEVPSYEF TSDDMVIVG
                           T  B BSS   S      EEE SSSSEEEE

                S = Bend
                T = Single Turn (minimum helix is defined at 2 n-turns)
                B = Single Bridge (ladders of length 1)
                G = 3₁₀-helix (3-helix)
                H = a-helix  (4-helix)
                S = disulfide bond
                E = extended ladders (b-strands)

Full DSSP:   DSSP text file link
 
 

2.    Class:    All beta proteins:  The ISP domain is in the class (secondary structure element) of all beta proteins indicating obviously that it is composed of all or mostly beta strands.  As in this case the Rieske ISP has 9 b-strands and only 1 a-helix.

3.    Fold:    ISP domain:  There were 104 members of the class of all beta proteins which share common core strucutres.  The ISP domain consists of two all-beta subdomains: conserved small domain has a rubredoxin-like fold; larger domain consists of 6 beta-strands packed in either sandwich of two 3-stranded sheets or closed barrel (n=6; S=8)

4.    Superfamily:     ISP domain(2): These two protein share a common structure and function
                                (1).  Rieske Iron-Sulfur Protein (electron transfer)
                                        Protein domains:  ISP subunit of the mitochondrial cytochrome bc1-complex,
                                        watersoluble domain with 3 species associated:  Cow (1RIE), Chicken, and Baker's Yeast.
                                (2).  Naphthalene 1,2-dioxygenase alpha subunit, N-domain (oxidoreductase)
                                        Species:  Pseudomonas putida

5.    Family:     Rieske iron-sulfur protein (ISP) (4):  These four proteins share a clear common evolutionary origin
                                (1).  Rieske Iron-Sulfur Protein (from bovine, gallus gallus, and saccharomyes cerevisiae)
                                     (2).  ISP subunit from chloroplast (spinacea oleracea)
                                     (3).  Arsenite oxidase Rieske subunit (alcaligenes faecalis)
                                (4). Rieske type ferredoxin associated with biphenyl dioxygenase (Burkholderia cepacia)

6.    Protein:    ISP subunit of the mitochondrial cytochrome bc1-complex, watersoluble domain

7.    Species:     Cow (Bos taurus)
 
 

There is only one relative of the Rieske ISP in the same fold group which has been calssified:  Rieske Soluble Fragment From Spinach.
Each share an overlap of 97/127 equivalent residues with a 76% overlap and a 22% sequence identity with a score of 83 out of 100.
 

Background Information:

Rieske Iron Sulfur Protein is a fragment of the ubiquitous cytochrome bc complexes which are involved in the electron transfer chains of bacteria, mitochondria, and choloroplasts.  "All bc complexes contain two heme b centres, heme c₁ or heme f and a 'Rieske' iron-sulfur protein comprising a high potential (300mV) [2Fe-2S] cluster."   Hydrophobic anchors link the ISP to the membrane part of the complex.  The iron-sulfur cluster is in the hydrophilic outer domain of the complex.  Further and more detailed information can be viewed at the following address:
 http://www.life.uiuc.edu/crofts/bc-complex_site/rieske_structure/rieske.html

The highlights of this paper are extrapolated below.

The above schematic ribbon diagrams (from above website), the left is a 90° rotation of the right with the disulfide bond removed.  This gives a better representation of the cluster involvoing the ligand residues.  The iron atoms are colored red while the sulfur atoms are colored yellow.  The distance between the two iron atoms is 2.7Å.

The residues coordinating the iron-sulfur cluster (2 cysteine and 2 histidine) can be viewed below:

The outer Iron atom is reduced by the quinol site, giving it a charge of +2, while the other iron atom retains a charge of +3.  The iron atoms and one of the sulfur atoms are buried within the protein and therefore are not solvent accessible.  The histidine ligands are fully exposed to the solvent.  Below (left) is an Electron Density Map of the active site of the Rieske ISP taken from http://www.csb.ki.se/xray/rieske.html.  The right displays a 2D structure of the active site.  Note that the histidine rings in the high resolution electron density map can be almost directly correlated and overlayed with the 2D structure.

The electron transfer takes place in the cytochrome b(6)f domain.  The active site of this protein is known as  the quinol oxidase site Q(0).  There are several confirmational changes that take place as this site is occupied and unoccupied and an intermediate stage between the two.  The conformational change is explained in two ways:
    1.  rotation of the entire extrinsic domain (from quinol site to cyt c1 subunit)
    2.  relative rotation between the cluster-binding fold and the base fold within the extrinsic domain

Further data showed that an electron is transfered from the quinol site to the ISP and then to the subunit cyt c₁.  This pathway has evolved to maximum efficiency and is important for energy conversion.

Sequence alignment:
In performing a sequence alignment, there were 6 entries that came up with 100% sequence alignment.  The Reiske Iron-Sulfur protein again, is a water soluble fragment of an entire complex, there were several entries which were the entire bc1 complex from different species as well from bovine.  Below is a representation of the Cytochrome Bc1 complex from bovine.  The lower right corner illustrates the iron-sulfur portion of this complex.
 
 

Structure Alignment from the Combinatorial Extension (CE):

     1RIE:_   3/71     AMS--KIE-IKLSD----IPEG--KNMAFKW-R--GKPLFVRHRTKKEIDQEAAVEVSQL
  1G8K:D    7/8     --P--ATAVSVAKN----LAAN--EPVSFTY-PDTSSPCVAVKLGA--------------
  1RFS:_   22/63    -------D-VIAAEWLKTHAPG--DRTLTQGLK--GDPTYLVVESD--------------
  1FQT:A    7/5     -----TRV-CDRRD----VPEG--EALKVES-G--GTSVAIFNVDG--------------
  1NDO:E   34/35    IFARNWLF-LTHDS----LIPAPGDYVTAKM-G--IDEVIVSRQ----ND----------
 

  1RIE:_   51/119   RDPQHDLERVKK--PEWV--ILIGVCTHLGCVP--I-----ANAGDFG--GYYCPCHGSH
  1G8K:D   42/43    -----PVPGGVGPDDDIV--AYSVLCTHMGCPT--S-----YDS-SSK--TFSCPCHFTE
  1RFS:_   56/97    --------------KTLATFGINAVCTHLGCVV--P-----FNAAE-N--KFICPCHGSQ
  1FQT:A   38/36    ---------------ELF--ATQDRCTHGDWSLSDG-----GYL---EGDVVECSLHMGK
  1NDO:E   72/73    --------------GSIR--AFLNVCRHRGKTL--VSVEAGNA----K--GFVCSYHGWG
 

  1RIE:_   98/166   YDAS--GRIRKGP------------APLNLEVPSYEFTS-DDMVIVG
  1G8K:D   85/86    FDAEKAGQMICGE------------ATADLPRVLLRYDAASDALTAV
  1RFS:_   92/133   YNNQ--GRVVRGP------------APLSLALAHCDVDD--GKVVFV
  1FQT:A   73/71    FCVRT-GKVKSPP------------PCEALKIFPIRI-E-DNDVLVD
  1NDO:E  108/109   FGSN--GELQSVPFEKDLYGESLNKKCLGLKEVARVESF-HGFIYGC
 
 
 

Note that the first 3 proteins in the above list share a common evolutionary origin with the Rieske Iron Sulfur Protein, while the last entry shares a common structure and function (SCOP).

ClustalW Multiple Sequence Alignment:

1RIE__       -----VLAMSKIEIKLSDIPEGKNMAFKWRGKPLFVRHRTKKEIDQEAAVEVSQLRDPQ- 54
1RFS__       -----------FVPPGGGAGTGGTIAKDALGNDVIAAEWLKTHAPGDRTLTQGLKGDPT- 48
1G8K_D       ------------RTTLAYPATAVSVAKNLAANEPVSFTYPDTSSP----CVAVKLGAPV- 43
1NDO_E       MNYNNKILVSESGLSQKHLIHGDEELFQHELKTIFARNWLFLTHDSLIPAPGDYVTAKMG 60
1FQT_A       ----GSHMKFTRVCDRRDVPEGEALKVESGGTSVAIFN---------------------- 34
                                  .     .   .                .

1RIE__       --HDLERVKKPEWVILIG-VCTHLG-CVPIANAGDFGGYYCPCHGSHYDAS--GRIRKGP 108
1RFS__       --YLVVESDKTLATFGINAVCTHLG-CVVPFNAAEN-KFICPCHGSQYNNQ--GRVVRGP 102
1G8K_D       --PGGVGPDDD--IVAYSVLCTHMG-CPTSYDSSSK-TFSCPCHFTEFDAEKAGQMICGE 97
1NDO_E       IDEVIVSRQNDGSIRAFLNVCRHRGKTLVSVEAGNAKGFGVCSYHGWFGSN--GELQSVP 118
1FQT_A       ---------VDGELFATQDRCTHGDWSLSDGGYLEGDVVECSLHMGKFCVR-TGKVKSPP 84
                     .           * * .         .     *. *   :     *.:

1RIE__       APLNLE--VPSYEFT-------------------------------------------SD 123
1RFS__       APLSLA--LAHCDVD-------------------------------------------DG 117
1G8K_D       ATADLPRVLLRYDAA-------------------------------------------SD 114
1NDO_E       FEKDLYGESLNKKCLGLKEVARVESFHGFIYGCFDQEAPPLMDYLGDAAWYLEPMFKHSG 178
1FQT_A       PCEALKIFPIRIEDN--------------------------------------------- 99
                 *       .                                             ..

1RIE__       DMVIVG------------------------------------------------------ 129
1RFS__       KVVFVPWTETDFRTGEAPWWSA-------------------------------------- 139
1G8K_D       ALTAVGVDGLIYGRQANVI----------------------------------------- 133
1NDO_E       GLELVGPPGKVVIKANWKAPAENFVGDAYHVGWTHASSLRSGESIFSSLAGNAALPPEGA 238
1FQT_A       -DVLVDFEAGYLAP---------------------------------------------- 112
                 *

In BOLD print are the residues and sequences that are conserved and aligned in both the combinatorial extension and in clustalw.

Each of these structures were aligned and downloaded.  Below is an image from Rasmol of the aligned structures.  Note the right portion of the protein.  The loops and b strands that are conserved in this area for each of the structures.  This is the active site of the protein (FeS) indicating a common function and similar structures.