Proc. Nat. Acad. Sci. USA
Vol. 72, No. 12, pp. 4854-4858, December 1975 Biochemistry
NH...S Hydrogen bonds in Peptococcus aerogenes ferredoxin, Clostridium pasteurianum rubredoxin, and Chromatium high potential iron protein (iron-sulfur protein/FeS cluster environment/protein conformation/isotope exchange/oxidation-reduction)
ELINOR ADMAN, KEITH D. WATENPAUGH, AND LYLE H. JENSEN Department of Biological Structure, University of Washington, School of Medicine, Seattle, Wash. 98195
Communicated by Edmond H. Fischer, September 24,1975
cluster formation and activity: It provides an environment which favors one form of iron-sulfur center over another (i.e., 1 Fe, 2 Fe-2 S, 4 Fe-4 S) and, in the case of the tetrameric clusters, it modulates the environments to stabilize one oxidation state or another, thereby modulating the operant redox potential. A notable feature of the arrangement of protein around the clusters in P. aerogenes ferredoxint and Clostridium pasteurianum rubredoxin (8) which may be involved in these functions is the number of NH..-S hydrogen bonds. They were also noted in the description of the structure of HiPIP (9), and the suggestion was made there that the existence of possible hydrogen bond donors in ferredoxin may contribute to the stabilization of a more negatively charged cluster. Indeed, P. aerogenes ferredoxin appears to have a disproportionate number of such bonds, having 15-18; HiPIP has 3-5, and rubredoxin has 6. NH-S bond geometry The NH-..S bonds, involving peptide nitrogen atoms and both inorganic and cysteine sulfur atoms, are illustrated in Figs. 1 and 2 for ferredoxin and rubedoxin, respectively. The N-S and H.--S distances are given in Tables 1, 2, and 3 for Fd, Rb, and HiPIP. N-S and H.--S distances are 3.25-3.55 A and 2.3-2.8 A, respectively, in small compounds (10). Since the errors associated with the protein coordinates are approximately 0.1-0.2 A, the protein N---S distances are in the range expected for hydrogen bonds. In three cases for Fd and one for Rb, distances are too large to describe as bonds; however, since the amide hydrogen atoms are directed toward the sulfur atoms they may be considered "incipient" hydrogen bonds. Several sulfur atoms have more than one amide H directed toward them. We observe four types of NH---S bonds, three involving cysteinyl sulfur atoms and one involving inorganic sulfur atoms. Two types use the cysteinyl sulfur atom of residue n and the peptide nitrogen of residue n + 2, and, in fact, are geometrically similar to Type I and Type II 31o NH--O bonds (11) as shown in Fig. 3 and described below. In a normal peptide 31o hydrogen bond the amide hydrogen atom of residue 4 (n + 3) is directed toward the carbonyl oxygen atom of residue 1 (n). The analogous NH---S geometry is achieved by rotating about the CaIC bond of residue 2 (n + 1) (Cys) so the amide hydrogen of residue 4 (n + 3) now points toward S'Y of residue 2 (n + 1). Since the carbonyl oxygen of residue 2 will eclipse a ,3 carbon on residue 3, depending on the orientation of the bridging peptide unit
ABSTRACT Results from refinement of the crystal structures of P. aerogenes ferredoxin and C pasteurianum rubredoxin determined by x-ray diffraction show that there are 15-18 NHOS bonds in the former and six in the latter with lengths in the range 3.1-3.9 A. Earlier tritium exchange experiments are consistent with the presence of these hydrogen onds in the ferredoxin structure and show that more peptide hydrogen atoms are available for exchange in apoferredoxin than in intact ferredoxin. Four types of NH-S bonds are observed and two of these are geometrically similar to the two. types of 310 NH-O bonds. The existence of more NH-S bonds in ferredoxin than in high potential iron protein suggests why the -2 form of the Fe4S4 cluster is preferred in ferredoxin over the -1 form found in high potential iron protein. From comparison of Cys-X-Y-Cys sequences in rubredoxin, terredoxin, and high potential iron protein we suggest that two Cys-X-Y-Cys-Z sequences, where Z may have conformation angles similar to glycine, are required to make a oneiron cluster, no more than one Cys-X-Y-Cys-Z-Gly sequence is required to form a Fe2S2 ferredoxin, and a Cys-X-Y-Cys-Gly sequence where Y has a conformation such that the cysteines bond to different iron atoms is necessary to form the tetrameric cluster.
The properties of iron-sulfur proteins raise an intriguing question: How does the protein structure contribute to the various observed forms of iron-sulfur centers? Iron-sulfur proteins include: (i) rubredoxin (Rb) with one Fe, no inorganic sulfur, and four cysteines; (ii) plant-type ferredoxins with two irons, two inorganic sulfur atoms, and at least four cysteines; and (iii) bacterial-type ferredoxin with one or more tetrameric clusters having four iron atoms, four inorganic sulfur atoms, and four cysteinyl sulfur atoms (1). The latter clusters are found both in Peptococcus aerogenes ferredoxin (Fd) (2) and in high potential iron protein (HiPIP) (3), a bacterial protein with unknown function which is reduced approximately +300 mV, in contrast to ferredoxin, which is reduced at the very low potential of about -400 mV. According to the three-state hypothesis (4), three oxidation states are accessible to the tetrameric cluster, one pair of which is preferred in HiPIP, another pair, of lower potential, in Fd. It has been demonstrated that the cluster in HiPIP can be "superreduced" in the presence of the denaturant dimethyl sulfoxide to the lowest state accessible to Fd (5), while the cluster in ferredoxin can be "superoxidized" in the presence of K3Fe(CN)6 (6). Studies with model compounds for tetrameric clusters have indicated that the -2/-3 reduction step analogous to reduction of ferredoxin operates at a much more negative potential than is observed for the proteins (7). Thus the protein portion of the iron-sulfur proteins has at least two functions that are important for
t
Abbreviations: Fd, ferredoxin; Rb, rubredoxin; HiPIP, high potential iron protein; S*, labile inorganic sulfide. 4854
E. Adman, L. C. Sieker, and L. H. Jensen, manuscript in preparation.
Biochemistry:
Adman et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
4855
FIG. 1. (a) Stereo drawing of NH...S bonds around the FeS cluster I of P. aerogenes ferredoxin. (b) Stereo drawing of NH...S bonds around FeS cluster II of P. aerogenes ferredoxin. Coordinates for the protein portion are idealized after refinement at 2.0 At. Single lines are drawn for N-H and H-S bonds.
in both the NH*..O and NH...S bonds, residue 3 must be a Gly in Type II NH...S bonds as well as in Type II NH...O bonds. We observe two Type II NH...S bonds in ferredoxin and two in rubredoxin, and in all cases residue 3 is a glycine. Type I and Type II bonds are each associated with characteristic conformational angles shown in Fig. 4. A third kind of NH...S bond involves a nitrogen distant in the sequence which completes a nearly tetrahedral coordination of atoms around a cysteinyl sulfur (see Tables 1-8). The sulfur atoms participating in this type of bond are StY, S3as, S'18, SY45, and in HiPIP, SY46. An even more distorted tetrahedral arrangement occurs at SY6 and S739 in rubredoxin (see Fig. 2). In the fourth kind, the N-H bond is directed toward inorganic sulfur atoms and is almost parallel to the Fe-S* bond distal to it, as if filling a position in a distorted octahedral arrangement of atoms around the S*.
Hydrogen isotope exchange studies The results of recent experiments with tritium exchange (12) on a homologous ferredoxin from C. acidi-urici taken in conjunction with our structural results indicate that the stability of NHI...S bonds may be similar to that observed for NH---O bonds. The tritium exchange results (observed at pH = 8.0) indicate about 16 amide hydrogen atoms (51 total amide hydrogen atoms minus 35 exchangeable ones) unavailable for exchange (that is, already participating in hydrogen bonding) in a reconstituted molecule, 27 in the oxidized form and 34 in the reduced form. In the structure of the oxidized form of P. aerogenes ferredoxin (crystallized at pH = 7.2) we observe eight main chain amide hydrogen bonds to oxygen atoms and 15 to sulfur, a total of 23, of 49 possible, which agrees remarkably well with the exchange
FIG. 2. Stereo drawing of NH...S bonds around FeS. cluster of C. pasteurianum rubredoxin. Coordinates for the protein portion are idealized after refinement at 1.5 A (8).
4856
Biochemistry: Adman et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
Table 1. NH ... S geometry in P. aerogenes ferredoxin* D- A
H--* A
(A)
(A)
..
Type I
D N10 N47 N37
N20t II III
IV
N13 N40 N28 N49 N2 N22 N9
N1lt N12
N14t N36 N38 N39 N41
A S8G S45G S35G S18G Sl1G S38G
S8G S45G S35G S18G S124 S124 S123 S234 S567 S678 S678 S578
3.6 3.7 3.3 > 4.0 3.6 3.4 3.4 3.6 3.5 3.9 3.7 > 4.0 3.1 3.9 3.7 3.3 3.5 3.1
2.7 2.7 2.4 3.0 2.7 2.5 2.4 2.6 2.5 2.9 2.8 3.4 2.4 3.4 2.8 2.5 2.8 2.4
Vol. 72, No. 12, pp. 4854-4858, December 1975 Biochemistry
NH...S Hydrogen bonds in Peptococcus aerogenes ferredoxin, Clostridium pasteurianum rubredoxin, and Chromatium high potential iron protein (iron-sulfur protein/FeS cluster environment/protein conformation/isotope exchange/oxidation-reduction)
ELINOR ADMAN, KEITH D. WATENPAUGH, AND LYLE H. JENSEN Department of Biological Structure, University of Washington, School of Medicine, Seattle, Wash. 98195
Communicated by Edmond H. Fischer, September 24,1975
cluster formation and activity: It provides an environment which favors one form of iron-sulfur center over another (i.e., 1 Fe, 2 Fe-2 S, 4 Fe-4 S) and, in the case of the tetrameric clusters, it modulates the environments to stabilize one oxidation state or another, thereby modulating the operant redox potential. A notable feature of the arrangement of protein around the clusters in P. aerogenes ferredoxint and Clostridium pasteurianum rubredoxin (8) which may be involved in these functions is the number of NH..-S hydrogen bonds. They were also noted in the description of the structure of HiPIP (9), and the suggestion was made there that the existence of possible hydrogen bond donors in ferredoxin may contribute to the stabilization of a more negatively charged cluster. Indeed, P. aerogenes ferredoxin appears to have a disproportionate number of such bonds, having 15-18; HiPIP has 3-5, and rubredoxin has 6. NH-S bond geometry The NH-..S bonds, involving peptide nitrogen atoms and both inorganic and cysteine sulfur atoms, are illustrated in Figs. 1 and 2 for ferredoxin and rubedoxin, respectively. The N-S and H.--S distances are given in Tables 1, 2, and 3 for Fd, Rb, and HiPIP. N-S and H.--S distances are 3.25-3.55 A and 2.3-2.8 A, respectively, in small compounds (10). Since the errors associated with the protein coordinates are approximately 0.1-0.2 A, the protein N---S distances are in the range expected for hydrogen bonds. In three cases for Fd and one for Rb, distances are too large to describe as bonds; however, since the amide hydrogen atoms are directed toward the sulfur atoms they may be considered "incipient" hydrogen bonds. Several sulfur atoms have more than one amide H directed toward them. We observe four types of NH---S bonds, three involving cysteinyl sulfur atoms and one involving inorganic sulfur atoms. Two types use the cysteinyl sulfur atom of residue n and the peptide nitrogen of residue n + 2, and, in fact, are geometrically similar to Type I and Type II 31o NH--O bonds (11) as shown in Fig. 3 and described below. In a normal peptide 31o hydrogen bond the amide hydrogen atom of residue 4 (n + 3) is directed toward the carbonyl oxygen atom of residue 1 (n). The analogous NH---S geometry is achieved by rotating about the CaIC bond of residue 2 (n + 1) (Cys) so the amide hydrogen of residue 4 (n + 3) now points toward S'Y of residue 2 (n + 1). Since the carbonyl oxygen of residue 2 will eclipse a ,3 carbon on residue 3, depending on the orientation of the bridging peptide unit
ABSTRACT Results from refinement of the crystal structures of P. aerogenes ferredoxin and C pasteurianum rubredoxin determined by x-ray diffraction show that there are 15-18 NHOS bonds in the former and six in the latter with lengths in the range 3.1-3.9 A. Earlier tritium exchange experiments are consistent with the presence of these hydrogen onds in the ferredoxin structure and show that more peptide hydrogen atoms are available for exchange in apoferredoxin than in intact ferredoxin. Four types of NH-S bonds are observed and two of these are geometrically similar to the two. types of 310 NH-O bonds. The existence of more NH-S bonds in ferredoxin than in high potential iron protein suggests why the -2 form of the Fe4S4 cluster is preferred in ferredoxin over the -1 form found in high potential iron protein. From comparison of Cys-X-Y-Cys sequences in rubredoxin, terredoxin, and high potential iron protein we suggest that two Cys-X-Y-Cys-Z sequences, where Z may have conformation angles similar to glycine, are required to make a oneiron cluster, no more than one Cys-X-Y-Cys-Z-Gly sequence is required to form a Fe2S2 ferredoxin, and a Cys-X-Y-Cys-Gly sequence where Y has a conformation such that the cysteines bond to different iron atoms is necessary to form the tetrameric cluster.
The properties of iron-sulfur proteins raise an intriguing question: How does the protein structure contribute to the various observed forms of iron-sulfur centers? Iron-sulfur proteins include: (i) rubredoxin (Rb) with one Fe, no inorganic sulfur, and four cysteines; (ii) plant-type ferredoxins with two irons, two inorganic sulfur atoms, and at least four cysteines; and (iii) bacterial-type ferredoxin with one or more tetrameric clusters having four iron atoms, four inorganic sulfur atoms, and four cysteinyl sulfur atoms (1). The latter clusters are found both in Peptococcus aerogenes ferredoxin (Fd) (2) and in high potential iron protein (HiPIP) (3), a bacterial protein with unknown function which is reduced approximately +300 mV, in contrast to ferredoxin, which is reduced at the very low potential of about -400 mV. According to the three-state hypothesis (4), three oxidation states are accessible to the tetrameric cluster, one pair of which is preferred in HiPIP, another pair, of lower potential, in Fd. It has been demonstrated that the cluster in HiPIP can be "superreduced" in the presence of the denaturant dimethyl sulfoxide to the lowest state accessible to Fd (5), while the cluster in ferredoxin can be "superoxidized" in the presence of K3Fe(CN)6 (6). Studies with model compounds for tetrameric clusters have indicated that the -2/-3 reduction step analogous to reduction of ferredoxin operates at a much more negative potential than is observed for the proteins (7). Thus the protein portion of the iron-sulfur proteins has at least two functions that are important for
t
Abbreviations: Fd, ferredoxin; Rb, rubredoxin; HiPIP, high potential iron protein; S*, labile inorganic sulfide. 4854
E. Adman, L. C. Sieker, and L. H. Jensen, manuscript in preparation.
Biochemistry:
Adman et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
4855
FIG. 1. (a) Stereo drawing of NH...S bonds around the FeS cluster I of P. aerogenes ferredoxin. (b) Stereo drawing of NH...S bonds around FeS cluster II of P. aerogenes ferredoxin. Coordinates for the protein portion are idealized after refinement at 2.0 At. Single lines are drawn for N-H and H-S bonds.
in both the NH*..O and NH...S bonds, residue 3 must be a Gly in Type II NH...S bonds as well as in Type II NH...O bonds. We observe two Type II NH...S bonds in ferredoxin and two in rubredoxin, and in all cases residue 3 is a glycine. Type I and Type II bonds are each associated with characteristic conformational angles shown in Fig. 4. A third kind of NH...S bond involves a nitrogen distant in the sequence which completes a nearly tetrahedral coordination of atoms around a cysteinyl sulfur (see Tables 1-8). The sulfur atoms participating in this type of bond are StY, S3as, S'18, SY45, and in HiPIP, SY46. An even more distorted tetrahedral arrangement occurs at SY6 and S739 in rubredoxin (see Fig. 2). In the fourth kind, the N-H bond is directed toward inorganic sulfur atoms and is almost parallel to the Fe-S* bond distal to it, as if filling a position in a distorted octahedral arrangement of atoms around the S*.
Hydrogen isotope exchange studies The results of recent experiments with tritium exchange (12) on a homologous ferredoxin from C. acidi-urici taken in conjunction with our structural results indicate that the stability of NHI...S bonds may be similar to that observed for NH---O bonds. The tritium exchange results (observed at pH = 8.0) indicate about 16 amide hydrogen atoms (51 total amide hydrogen atoms minus 35 exchangeable ones) unavailable for exchange (that is, already participating in hydrogen bonding) in a reconstituted molecule, 27 in the oxidized form and 34 in the reduced form. In the structure of the oxidized form of P. aerogenes ferredoxin (crystallized at pH = 7.2) we observe eight main chain amide hydrogen bonds to oxygen atoms and 15 to sulfur, a total of 23, of 49 possible, which agrees remarkably well with the exchange
FIG. 2. Stereo drawing of NH...S bonds around FeS. cluster of C. pasteurianum rubredoxin. Coordinates for the protein portion are idealized after refinement at 1.5 A (8).
4856
Biochemistry: Adman et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
Table 1. NH ... S geometry in P. aerogenes ferredoxin* D- A
H--* A
(A)
(A)
..
Type I
D N10 N47 N37
N20t II III
IV
N13 N40 N28 N49 N2 N22 N9
N1lt N12
N14t N36 N38 N39 N41
A S8G S45G S35G S18G Sl1G S38G
S8G S45G S35G S18G S124 S124 S123 S234 S567 S678 S678 S578
3.6 3.7 3.3 > 4.0 3.6 3.4 3.4 3.6 3.5 3.9 3.7 > 4.0 3.1 3.9 3.7 3.3 3.5 3.1
2.7 2.7 2.4 3.0 2.7 2.5 2.4 2.6 2.5 2.9 2.8 3.4 2.4 3.4 2.8 2.5 2.8 2.4
