Escherichia coli flavohaemoglobin (Hmp) reduces cytochrome c and Fe(III)-hydroxamate K by electron transfer from NADH via FAD: sensitivity of oxidoreductase activity to haem-bound dioxygen

Abstract

JapanEscherichia coli flavohaemoglobin (Hmp) reduced purified mitochondria1 cytochrome c aerobically in a reaction that was not substantially inhibited by superoxide dismutase, demonstrating that superoxide anion, the product of 0, reduction by Hmp, did not contribute markedly to cytochrome c reduction. Cytochrome c was reduced by Hmp even in the presence of 0 5 mM CO, when the haem B was locked in the ferrous, low-spin state, demonstrating that electron transfer to cytochrome c from NADH was via FAD, not haem. Hmp also reduced the ferrisiderophore complex Fe(lll)-hydroxamate K from Rhizobium leguminosanrm bv. wiciae anaerobically in a CO-insensitive manner, but at low rates and with low affinity for this substrate. The NADH+ytochrome c oxidoreductase activity of Hmp was slightly sensitive to the binding and reduction of 0, at the haem. The V-of cytochrome c reduction fell from 7.1 s-l in the presence of 0 5 mM CO to 5 0 s'l in the presence of 100 pM O,, with no significant change in Km for cytochrome c (68 to 7.3 pM, respectively). 0, at near-micromolar concentrations diminished cytochrome c reduction to a similar extent as did 100 pM 0 , . Thus, Hmp acts as a reductase of broad specificity, apparently without involvement of electron transfer via the globinlike haem. These data are consistent with the hypothesis that Hmp could act as an intracellular sensor of 0, since, in the absence of O, , electron flux from FAD to other electron acceptors increases. However, the nature of such acceptors in wivo is not known and alternative models for 0, sensing are also considered.

Paraquat regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12 is SoxRS independent but modulated by sigma S

“…First, use of a ⌽(sodA-lacZ) fusion to monitor intracellular superoxide production demonstrated that Hmp is capable of generating superoxide in vivo when overexpressed (30). Second, oxygen is bound at a single heme in Hmp which accepts electrons from NADH via flavin adenine dinucleotide (18,37,39), facilitating single-electron reduction of bound O 2 to superoxide anion. Indeed, studies of purified Hmp in vitro have demonstrated that Hmp generates superoxide which reduces Fe(III) to Fe(II) (30), although the reduction of other substrates (e.g., cytochrome c) by Hmp is not superoxide dependent (39).…”

“…Second, oxygen is bound at a single heme in Hmp which accepts electrons from NADH via flavin adenine dinucleotide (18,37,39), facilitating single-electron reduction of bound O 2 to superoxide anion. Indeed, studies of purified Hmp in vitro have demonstrated that Hmp generates superoxide which reduces Fe(III) to Fe(II) (30), although the reduction of other substrates (e.g., cytochrome c) by Hmp is not superoxide dependent (39). Third, ⌽(hmp-lacZ) expression is regulated in a complex manner (38).…”

“…Hmp binds oxygen to the high-spin ferrous heme to form an oxygenated complex (18). Previous work in our laboratory has attempted to define the function(s) of Hmp by studying how hmp expression is regulated (31,38) and the catalytic capabilities of Hmp (30,39). The expression of hmp is strongly induced by nitrite and nitric oxide, suggesting that Hmp may be involved in the metabolism of these compounds (38) or is responsive to oxidative stress.…”

“…The expression of hmp is strongly induced by nitrite and nitric oxide, suggesting that Hmp may be involved in the metabolism of these compounds (38) or is responsive to oxidative stress. Other evidence demonstrates that Hmp contributes to oxidative stress in vivo by producing superoxide anion (30,39).…”

Steady-state and Transient Kinetics of Escherichia coli Nitric-oxide Dioxygenase (Flavohemoglobin)

“…Moreover, there is evidence for a separate NOinducible NO reductase activity in E. coli that performs this function (14). Other activities of flavoHb have been described (45)(46)(47), but their biological significance remains to be determined.…”