Azotobacter vinelandii ferredoxin I. Aspartate 15 facilitates proton transfer to the reduced [3Fe-4S] cluster

B. Shen, L. L. Martin, J. N. Butt, F. A. Armstrong, C. D. Stout, G. M. Jensen, P. J. Stephens, G. N. La Mar, C. M. Gorst, B. K. Burgess

Research output: Contribution to journalArticleResearchpeer-review

90 Citations (Scopus)

Abstract

The [3Fe-4S](+/0) cluster of Azotobacter vinelandii ferredoxin I (AvFdI) has an unusually low and strongly pH-dependent reduction potential (E'0). The reduced cluster exists in two forms, depending upon pH, that exhibit substantially different magnetic circular dichroism (MCD) spectra. Recent studies have established that the MCD changes observed on decreasing the pH from 8.3 (alkaline form) to 6.0 (acid form) cannot be explained either by a change in spin state of the cluster (Stephens, P. J., Jensen, G. M., Devlin, F. J., Morgan, T. V., Stout, C. D., Martin, A. E., and Burgess, B. K. (1991) Biochemistry 30, 3200-3209) or by a major structural change (e.g. ligand exchange) (Stout, C. D. (1993) J. Biol. Chem. 268, 25920-25927). Here, we have examined the influence of aspartate 15 on the pH dependence of the spectroscopic and electrochemical properties of AvFdI by construction of a D15N mutant. Aspartate 15, which is salt-bridged to lysine 84 at the protein surface, is the closest ionizable residue to the [3Fe-4S] cluster. The results show that replacement of aspartate by asparagine results in an ~20- mV increase in E'0 for the [3Fe-4S](+/0) cluster at high pH concomitant with an ~0.8-pH unit decrease in the pK of the reduced form. The major pH dependence of E'0 is preserved as is the effect observed by MCD. These data eliminate the possibility that the MCD change is due to the presence of Asp- 15 and support the conclusion that it originates in direct protonation of the [3Fe-4S]0 cluster, probably on a sulfide ion. Voltammetric studies show that interconversion between [3Fe-4S]+ and [3Fe-4S]0 at acidic pH involves rapid electron transfer followed by proton transfer (for reduction) and then proton transfer followed by electron transfer (for oxidation). Ionized aspartate 15 facilitates proton transfer. Thus, protonation and deprotonation are much slower for D15N relative to the native protein at pH >5.5. Proton transfer reactions necessary for further reduction of the [3Fe-4S]0 cluster to the [3Fe-4S]- and [3Fe-4S]2- states are also retarded in D15N. The results suggest that the carboxylate-ammonium salt bridge afforded by Asp-15-Lys-84 conducts protons between the cluster and solvent H2O molecules. Overproduction of D15N FdI, but not native FdI, in A. vinelandii has a negative effect on the growth rate of the organism, suggesting that the rate of protonation or deprotonation of the [3Fe-4S]0 cluster may be important in vivo.

Original languageEnglish
Pages (from-to)25928-25939
Number of pages12
JournalJournal of Biological Chemistry
Volume268
Issue number34
Publication statusPublished - 1 Jan 1993
Externally publishedYes

Cite this

Shen, B., Martin, L. L., Butt, J. N., Armstrong, F. A., Stout, C. D., Jensen, G. M., ... Burgess, B. K. (1993). Azotobacter vinelandii ferredoxin I. Aspartate 15 facilitates proton transfer to the reduced [3Fe-4S] cluster. Journal of Biological Chemistry, 268(34), 25928-25939.
Shen, B. ; Martin, L. L. ; Butt, J. N. ; Armstrong, F. A. ; Stout, C. D. ; Jensen, G. M. ; Stephens, P. J. ; La Mar, G. N. ; Gorst, C. M. ; Burgess, B. K. / Azotobacter vinelandii ferredoxin I. Aspartate 15 facilitates proton transfer to the reduced [3Fe-4S] cluster. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 34. pp. 25928-25939.
@article{18438ff906da4d6e9e61397b7b2c8fde,
title = "Azotobacter vinelandii ferredoxin I. Aspartate 15 facilitates proton transfer to the reduced [3Fe-4S] cluster",
abstract = "The [3Fe-4S](+/0) cluster of Azotobacter vinelandii ferredoxin I (AvFdI) has an unusually low and strongly pH-dependent reduction potential (E'0). The reduced cluster exists in two forms, depending upon pH, that exhibit substantially different magnetic circular dichroism (MCD) spectra. Recent studies have established that the MCD changes observed on decreasing the pH from 8.3 (alkaline form) to 6.0 (acid form) cannot be explained either by a change in spin state of the cluster (Stephens, P. J., Jensen, G. M., Devlin, F. J., Morgan, T. V., Stout, C. D., Martin, A. E., and Burgess, B. K. (1991) Biochemistry 30, 3200-3209) or by a major structural change (e.g. ligand exchange) (Stout, C. D. (1993) J. Biol. Chem. 268, 25920-25927). Here, we have examined the influence of aspartate 15 on the pH dependence of the spectroscopic and electrochemical properties of AvFdI by construction of a D15N mutant. Aspartate 15, which is salt-bridged to lysine 84 at the protein surface, is the closest ionizable residue to the [3Fe-4S] cluster. The results show that replacement of aspartate by asparagine results in an ~20- mV increase in E'0 for the [3Fe-4S](+/0) cluster at high pH concomitant with an ~0.8-pH unit decrease in the pK of the reduced form. The major pH dependence of E'0 is preserved as is the effect observed by MCD. These data eliminate the possibility that the MCD change is due to the presence of Asp- 15 and support the conclusion that it originates in direct protonation of the [3Fe-4S]0 cluster, probably on a sulfide ion. Voltammetric studies show that interconversion between [3Fe-4S]+ and [3Fe-4S]0 at acidic pH involves rapid electron transfer followed by proton transfer (for reduction) and then proton transfer followed by electron transfer (for oxidation). Ionized aspartate 15 facilitates proton transfer. Thus, protonation and deprotonation are much slower for D15N relative to the native protein at pH >5.5. Proton transfer reactions necessary for further reduction of the [3Fe-4S]0 cluster to the [3Fe-4S]- and [3Fe-4S]2- states are also retarded in D15N. The results suggest that the carboxylate-ammonium salt bridge afforded by Asp-15-Lys-84 conducts protons between the cluster and solvent H2O molecules. Overproduction of D15N FdI, but not native FdI, in A. vinelandii has a negative effect on the growth rate of the organism, suggesting that the rate of protonation or deprotonation of the [3Fe-4S]0 cluster may be important in vivo.",
author = "B. Shen and Martin, {L. L.} and Butt, {J. N.} and Armstrong, {F. A.} and Stout, {C. D.} and Jensen, {G. M.} and Stephens, {P. J.} and {La Mar}, {G. N.} and Gorst, {C. M.} and Burgess, {B. K.}",
year = "1993",
month = "1",
day = "1",
language = "English",
volume = "268",
pages = "25928--25939",
journal = "Journal of Biological Chemistry",
issn = "1083-351X",
publisher = "American Society for Biochemistry and Molecular Biology",
number = "34",

}

Shen, B, Martin, LL, Butt, JN, Armstrong, FA, Stout, CD, Jensen, GM, Stephens, PJ, La Mar, GN, Gorst, CM & Burgess, BK 1993, 'Azotobacter vinelandii ferredoxin I. Aspartate 15 facilitates proton transfer to the reduced [3Fe-4S] cluster', Journal of Biological Chemistry, vol. 268, no. 34, pp. 25928-25939.

Azotobacter vinelandii ferredoxin I. Aspartate 15 facilitates proton transfer to the reduced [3Fe-4S] cluster. / Shen, B.; Martin, L. L.; Butt, J. N.; Armstrong, F. A.; Stout, C. D.; Jensen, G. M.; Stephens, P. J.; La Mar, G. N.; Gorst, C. M.; Burgess, B. K.

In: Journal of Biological Chemistry, Vol. 268, No. 34, 01.01.1993, p. 25928-25939.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Azotobacter vinelandii ferredoxin I. Aspartate 15 facilitates proton transfer to the reduced [3Fe-4S] cluster

AU - Shen, B.

AU - Martin, L. L.

AU - Butt, J. N.

AU - Armstrong, F. A.

AU - Stout, C. D.

AU - Jensen, G. M.

AU - Stephens, P. J.

AU - La Mar, G. N.

AU - Gorst, C. M.

AU - Burgess, B. K.

PY - 1993/1/1

Y1 - 1993/1/1

N2 - The [3Fe-4S](+/0) cluster of Azotobacter vinelandii ferredoxin I (AvFdI) has an unusually low and strongly pH-dependent reduction potential (E'0). The reduced cluster exists in two forms, depending upon pH, that exhibit substantially different magnetic circular dichroism (MCD) spectra. Recent studies have established that the MCD changes observed on decreasing the pH from 8.3 (alkaline form) to 6.0 (acid form) cannot be explained either by a change in spin state of the cluster (Stephens, P. J., Jensen, G. M., Devlin, F. J., Morgan, T. V., Stout, C. D., Martin, A. E., and Burgess, B. K. (1991) Biochemistry 30, 3200-3209) or by a major structural change (e.g. ligand exchange) (Stout, C. D. (1993) J. Biol. Chem. 268, 25920-25927). Here, we have examined the influence of aspartate 15 on the pH dependence of the spectroscopic and electrochemical properties of AvFdI by construction of a D15N mutant. Aspartate 15, which is salt-bridged to lysine 84 at the protein surface, is the closest ionizable residue to the [3Fe-4S] cluster. The results show that replacement of aspartate by asparagine results in an ~20- mV increase in E'0 for the [3Fe-4S](+/0) cluster at high pH concomitant with an ~0.8-pH unit decrease in the pK of the reduced form. The major pH dependence of E'0 is preserved as is the effect observed by MCD. These data eliminate the possibility that the MCD change is due to the presence of Asp- 15 and support the conclusion that it originates in direct protonation of the [3Fe-4S]0 cluster, probably on a sulfide ion. Voltammetric studies show that interconversion between [3Fe-4S]+ and [3Fe-4S]0 at acidic pH involves rapid electron transfer followed by proton transfer (for reduction) and then proton transfer followed by electron transfer (for oxidation). Ionized aspartate 15 facilitates proton transfer. Thus, protonation and deprotonation are much slower for D15N relative to the native protein at pH >5.5. Proton transfer reactions necessary for further reduction of the [3Fe-4S]0 cluster to the [3Fe-4S]- and [3Fe-4S]2- states are also retarded in D15N. The results suggest that the carboxylate-ammonium salt bridge afforded by Asp-15-Lys-84 conducts protons between the cluster and solvent H2O molecules. Overproduction of D15N FdI, but not native FdI, in A. vinelandii has a negative effect on the growth rate of the organism, suggesting that the rate of protonation or deprotonation of the [3Fe-4S]0 cluster may be important in vivo.

AB - The [3Fe-4S](+/0) cluster of Azotobacter vinelandii ferredoxin I (AvFdI) has an unusually low and strongly pH-dependent reduction potential (E'0). The reduced cluster exists in two forms, depending upon pH, that exhibit substantially different magnetic circular dichroism (MCD) spectra. Recent studies have established that the MCD changes observed on decreasing the pH from 8.3 (alkaline form) to 6.0 (acid form) cannot be explained either by a change in spin state of the cluster (Stephens, P. J., Jensen, G. M., Devlin, F. J., Morgan, T. V., Stout, C. D., Martin, A. E., and Burgess, B. K. (1991) Biochemistry 30, 3200-3209) or by a major structural change (e.g. ligand exchange) (Stout, C. D. (1993) J. Biol. Chem. 268, 25920-25927). Here, we have examined the influence of aspartate 15 on the pH dependence of the spectroscopic and electrochemical properties of AvFdI by construction of a D15N mutant. Aspartate 15, which is salt-bridged to lysine 84 at the protein surface, is the closest ionizable residue to the [3Fe-4S] cluster. The results show that replacement of aspartate by asparagine results in an ~20- mV increase in E'0 for the [3Fe-4S](+/0) cluster at high pH concomitant with an ~0.8-pH unit decrease in the pK of the reduced form. The major pH dependence of E'0 is preserved as is the effect observed by MCD. These data eliminate the possibility that the MCD change is due to the presence of Asp- 15 and support the conclusion that it originates in direct protonation of the [3Fe-4S]0 cluster, probably on a sulfide ion. Voltammetric studies show that interconversion between [3Fe-4S]+ and [3Fe-4S]0 at acidic pH involves rapid electron transfer followed by proton transfer (for reduction) and then proton transfer followed by electron transfer (for oxidation). Ionized aspartate 15 facilitates proton transfer. Thus, protonation and deprotonation are much slower for D15N relative to the native protein at pH >5.5. Proton transfer reactions necessary for further reduction of the [3Fe-4S]0 cluster to the [3Fe-4S]- and [3Fe-4S]2- states are also retarded in D15N. The results suggest that the carboxylate-ammonium salt bridge afforded by Asp-15-Lys-84 conducts protons between the cluster and solvent H2O molecules. Overproduction of D15N FdI, but not native FdI, in A. vinelandii has a negative effect on the growth rate of the organism, suggesting that the rate of protonation or deprotonation of the [3Fe-4S]0 cluster may be important in vivo.

UR - http://www.scopus.com/inward/record.url?scp=0027518193&partnerID=8YFLogxK

M3 - Article

C2 - 8245026

AN - SCOPUS:0027518193

VL - 268

SP - 25928

EP - 25939

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 1083-351X

IS - 34

ER -