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Title: Cyanide inactivation of hydrogenase from Azotobacter vinelandii

Abstract

The effects of cyanide on membrane-associated and purified hydrogenase from Azotobacter vinelandii were characterized. Inactivation of hydrogenase by cyanide was dependent on the activity (oxidation) state of the enzyme. Active (reduced) hydrogenase showed no inactivation when treated with cyanide over several hours. Treatment of reversibly inactive (oxidized) states of both membrane-associated and purified hydrogenase, however, resulted in a time-dependent, irreversible loss of hydrogenase activity. The rate of cyanide inactivation was dependent on the cyanide concentration and was an apparent first-order process for purified enzyme (bimolecular rate constant, 23.1 M{sup {minus}1} min{sup {minus}1} for CN{sup {minus}}). The rate of inactivation decreased with decreasing pH. ({sup 14}C)cyanide remained associated with cyanide-inactivated hydrogenase after gel filtration chromatography, with a stoichiometry of 1.7 mol of cyanide bound per mol of inactive enzyme. The presence of saturating concentrations of CO had no effect on the rate or extent of cyanide inactivation of hydrogenases. The results indicate that cyanide can cause a time-dependent, irreversible inactivation of hydrogenase in the oxidized, activatable state but has no effect when hydrogenase is in the reduced, active state.

Authors:
;  [1]
  1. (Univ. of California, Riverside (USA))
Publication Date:
OSTI Identifier:
6936059
DOE Contract Number:
FG03-84ER13257
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Bacteriology; (USA); Journal Volume: 171:6
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; 59 BASIC BIOLOGICAL SCIENCES; AZOTOBACTER; SENSITIVITY; CYANIDES; TOXICITY; HYDROGENASES; INACTIVATION; CARBON 14 COMPOUNDS; ENZYME ACTIVITY; TRACER TECHNIQUES; BACTERIA; ENZYMES; ISOTOPE APPLICATIONS; LABELLED COMPOUNDS; MICROORGANISMS; OXIDOREDUCTASES 560300* -- Chemicals Metabolism & Toxicology; 550201 -- Biochemistry-- Tracer Techniques

Citation Formats

Seefeldt, L.C., and Arp, D.J.. Cyanide inactivation of hydrogenase from Azotobacter vinelandii. United States: N. p., 1989. Web.
Seefeldt, L.C., & Arp, D.J.. Cyanide inactivation of hydrogenase from Azotobacter vinelandii. United States.
Seefeldt, L.C., and Arp, D.J.. 1989. "Cyanide inactivation of hydrogenase from Azotobacter vinelandii". United States. doi:.
@article{osti_6936059,
title = {Cyanide inactivation of hydrogenase from Azotobacter vinelandii},
author = {Seefeldt, L.C. and Arp, D.J.},
abstractNote = {The effects of cyanide on membrane-associated and purified hydrogenase from Azotobacter vinelandii were characterized. Inactivation of hydrogenase by cyanide was dependent on the activity (oxidation) state of the enzyme. Active (reduced) hydrogenase showed no inactivation when treated with cyanide over several hours. Treatment of reversibly inactive (oxidized) states of both membrane-associated and purified hydrogenase, however, resulted in a time-dependent, irreversible loss of hydrogenase activity. The rate of cyanide inactivation was dependent on the cyanide concentration and was an apparent first-order process for purified enzyme (bimolecular rate constant, 23.1 M{sup {minus}1} min{sup {minus}1} for CN{sup {minus}}). The rate of inactivation decreased with decreasing pH. ({sup 14}C)cyanide remained associated with cyanide-inactivated hydrogenase after gel filtration chromatography, with a stoichiometry of 1.7 mol of cyanide bound per mol of inactive enzyme. The presence of saturating concentrations of CO had no effect on the rate or extent of cyanide inactivation of hydrogenases. The results indicate that cyanide can cause a time-dependent, irreversible inactivation of hydrogenase in the oxidized, activatable state but has no effect when hydrogenase is in the reduced, active state.},
doi = {},
journal = {Journal of Bacteriology; (USA)},
number = ,
volume = 171:6,
place = {United States},
year = 1989,
month = 6
}
  • The effects of O{sub 2} on membrane-bound Azotobacter vinelandii hydrogenase in both the membrane-associated and purified states were characterized. O{sub 2} was a rapid-equilibrium, reversible inhibitor both of H{sub 2} oxidation/electron acceptor reduction activity and of the exchange activity. O{sub 2} inhibition was noncompetitive versus the electron acceptor methylene blue and uncompetitive versus the substrate H{sub 2}. O{sub 2} inhibition was rapidly reversed by removal of the O{sub 2}. In addition to an O{sub 2}-independent, reversible inhibition of hydrogenase, a time-dependent, irreversible inactivation by O{sub 2} of both H{sub 2} oxidation and exchange activities for this hydrogenase was demonstrated. Themore » irreversible inactivation followed a first-order process. The inactivation by O{sub 2} was protected against by the substrate H{sub 2} in a concentration-dependent manner. This is the first report of H{sub 2} protection from O{sub 2}-dependent, irreversible inactivation for any hydrogenase. The competitive inhibitor CO did not affect the irreversible inactivation by O{sub 2} or the protection by H{sub 2}. A model is proposed that outlines various oxidation states of this hydrogenase and the effects of O{sub 2} on each state.« less
  • The inhibition of purified and membrane-bound hydrogenase from Azotobacter vinelandii by dihydrogen-free acetylene was investigated. The inhibition was a time-dependent process which exhibited first-order kinetics. Both H/sub 2/ and CO protected against the inhibition by acetylene. K/sub protect(app)/ values of 0.41 and 24 ..mu..M were derived for these gases, respectively. Both H/sub 2/-oxidizing activity and the tritium exchange capacity of the purified enzyme were inhibited at the same rate by acetylene. Removal of acetylene reversed the inhibition for both the purified and the membrane-associated form of the enzyme. The purified hydrogenases from both Rhizobium japonicum and Alcaligenes eutrophus H16 weremore » also inhibited by acetylene in a time-dependent fashion. These findings suggest that acetylene is an active-site-directed, slow-binding, reversible inhibitor of some membrane-bound hydrogenases from aerobic bacteria.« less
  • Acetylene is a slow-binding inhibitor of the Ni- and Fe-containing dimeric hydrogenase isolated from Azotobacter vinelandii. Acetylene was released from hydrogenase during the recovery from inhibition. This indicates that no transformation of acetylene to another compound occurred as a result of the interaction with hydrogenase. However, the release of C{sub 2}H{sub 2} proceeds more rapidly than the recovery of activity, which indicates that release of C{sub 2}H{sub 2} is not sufficient for recovery of activity. Acetylene binds tightly to native hydrogenase; hydrogenase and radioactivity coelute from a gel permeation column following inhibition with {sup 14}C{sub 2}H{sub 2}. Acetylene, or amore » derivative, remains bound to the large 65,000 MW subunit (and not to the small 35,000 MW subunit) of hydrogenase following denaturation as evidence by SDS-PAGE and fluorography of {sup 14}C{sub 2}H{sub 2}-inhibited hydrogenase. This result suggests that C{sub 2}H{sub 2}, and by analogy H{sub 2}, binds to and is activated by the large subunit of this dimeric hydrogenase. Radioactivity is lost from {sup 14}C{sub 2}H{sub 2}-inhibited protein during recovery. The inhibition is remarkably specific for C{sub 2}H{sub 2}; propyne, butyne, and ethylene are not inhibitors.« less
  • The biological reduction of dinitrogen (N 2) to ammonia (NH 3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (E m = -320 mV) coupled tomore » reduction of flavodoxin semiquinone (E m = -460 mV) and reduction of coenzyme Q (E m = 10 mV). Knocking out fix genes rendered ..delta..rnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Altogether, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.« less