Respiratory Enzymes of Thiobacillus ferrooxidans. Kinetic Properties of an Acid-Stable Iron:Rusticyanin Oxidoreductase

Abstract

Rusticyanin is an acid-stable, soluble blue copper protein found in abundance in the periplasmic space of Thiobacillus ferrooxidans, an acidophilic bacterium capable of growing autotrophically on soluble ferrous sulfate. An acid-stable iron:rusticyanin oxidoreductase activity was partially purified from cell-free extracts of T. ferrooxidans. The enzyme-catalyzed, iron-dependent reduction of the rusticyanin exhibited three kinetic properties characteristic of aerobic iron oxidation by whole cells. (i) A survey of 14 different anions indicated that catalysis by the oxidoreductase occurred only in the presence of sulfate or selenate, an anion specificity identical to that of whole cells. (ii) Saturation with both sulfatoiron(II) and the catalyst produced a concentration-independent rate constant of 3 s-1 for the reduction of the rusticyanin, which is an electron transfer reaction sufficiently rapid to account for the flux of electrons through the iron respiratory chain. (iii) Values for the enzyme-catalyzed pseudo-first-order rate constants for the reduction of the rusticyanin showed a hyperbolic dependence on the concentration of sulfatoiron(II) with a half-maximal effect at 300 microM, a value similar to the apparent KM for iron shown by whole cells. On the basis of these favorable comparisons between the behavior patterns of isolated biomolecules and those of whole cells, this iron:rusticyanin oxidoreductase is postulated to be the primary cellular oxidant of ferrous ions in the iron respiratory electron transport chain of T. ferrooxidans.

The Multicenter Aerobic Iron Respiratory Chain of Acidithiobacillus ferrooxidans Functions as an Ensemble with a Single Macroscopic Rate Constant

“…6B, where the concentrations of respiratory proteins in the periplasm are orders of magnitude higher than any encountered in published in vitro functional studies. Studies conducted in dilute solutions have shown the following: (i) rusticyanin forms a complex with the periplasmic cytochrome c that lowers the standard reduction potential of the blue protein (14), and (ii) the transfer of electrons from soluble Fe(II) to rusticyanin is catalyzed by the outer membrane cytochrome c via a transient encounter-Michaelis complex (7,12). It is not a stretch of the imagination to hypothesize that additional interactions that serve to further influence their function and reactivity occur among the respiratory proteins at these high concentrations in the periplasm.…”

“…A number of laboratories have described redox-active proteins expressed by At. ferrooxidans that might participate in the iron respiratory chain (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Perhaps the most generally accepted model for the iron respiratory chain of At.…”

“…The rus operon is hypothesized to encode all of the principal electron transport proteins that compose the iron respiratory chain (15). The primary oxidation of extracellular ferrous iron is thought to be accomplished by an outer membrane cytochrome c (7,12) that passes the electrons to rusticyanin, a type I blue copper protein located in the periplasmic space of this Gram-negative organism (1,24). These electrons are then transferred from the rusticyanin via a different periplasmic cytochrome c (14) to an aa 3 -type terminal oxidase that spans the cytoplasmic membrane and transfers electrons to molecular oxygen (6,13).…”

Specific iron oxidation and cell growth rates of bacteria in batch culture

“…Sulfate is known to bind preferentially to Fe 3+ relative to Fe 2+ , the effect is to significantly reduce E III/II couple's E° value (Table 1). The external electron transport chain between the Fe(III/II) and O 2 /H 2 O couples has been intensively studied (Rohwerder et al, 2003;Ingledew and Cobley, 1980;Shute 1987 and1994;Brasseur et al, 2004; M A N U S C R I P T ACCEPTED MANUSCRIPT Elbehti et al, 1999 and2000;Malarte et al, 2005) and redox active components have been characterised. These redox mediators appear to have either iron (Castelle et al, 2008) or copper as their redox active centre (Ida et al, 2003;McGinnis et al, 1986;Kyritsis et al, 1995;Giudici-Orticoni et al, 1999 Our interpretation of (dII/dt)/N patterns observed in Fig.…”

Respiratory Isozyme, Two Types of Rusticyanin ofAcidithiobacillus ferrooxidans

“…[1][2][3] Aerobic respiration on iron is the principle metabolic activity of the chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans, which obtains all of its energy for CO2ˆxation and growth from oxidation of ferrous ions. [4][5][6][7][8][9][10] In that process, A. ferrooxidans uses rusticyanin as an electron transfer protein within its oxidative respiratory chain. 4,5,[10][11][12][13][14][15] This type I protein belongs to the copper protein superfamily the members of which are widely distributed in numerous organisms, 13,16) however, rusticyanin is the only member known to have both the high redox potential (＋680 mV) necessary for an electron carrier and acid stability at pH 2-3 where A. ferrooxidans thrives.…”