Long-range interactions between the Fe protein binding sites of the MoFe protein of nitrogenase

Abstract

We report the properties and reactivity of the catalytically active heterologous nitrogenase formed between the Fe protein from Clostridium pasteurianum (Cp2) and the MoFe protein from Klebsiella pneumoniae (Kp1). Under turnover conditions, in the presence of MgATP, a stable 2:1 (Cp2)2Kp1 electron transfer complex is formed, in which the [4Fe-4S]+ centre of Cp2 is protected from chelation by alpha,alpha'-bipyridyl. However, the two Fe protein-binding sites on Kp1 are not equivalent, since a 1:1 Cp2.Kp1 complex was isolated by gel filtration. The non-equivalence of the Fe protein binding sites was also indicated by the inhibition pattern of Klebsiella nitrogenase by Cp2. The EPR spectrum of the isolated 1:1 Cp2.Kp1 complex showed an S=1/2 signal characteristic of dithionite-reduced Cp2 and signals with g values of 4.27, 3.73, 2.01 and 4.32, 3.63, 2.00 characteristic of the high- and low-pH forms of the FeMoco centre of Kp1, respectively. The unoccupied binding site of Kp1 of the isolated 1:1 Cp2Kp1 complex was shown to be catalytically fully functional in combination with Kp2. In contrast to homologous nitrogenases, which require MgATP for detectable rates of electron transfer from the Fe protein, stopped-flow kinetic studies revealed that electron transfer from Cp2 to Kp1 occurred in the absence of MgATP with a rate constant of 0.065 s(-1). Subsequently, a slower transient decrease and restoration of absorption in the electronic spectrum in the 500-700 nm region was observed. These changes corresponded with those in the intensity of the S=3/2 EPR signal of the FeMoco centres of Kp1 and were consistent with the transient reduction of the FeMoco centre of Kp1 to an EPR-silent form, followed by restoration of the signal at longer reaction times. These changes were not associated with catalysis since no evolution of H2 was detectable.

Novel EPR signals associated with FeMoco centres of MoFe protein in MgADP‐inhibited turnover of nitrogenase

“…Cp2 remains oxidised at prolonged reaction times, even in the presence of excess dithionite. It has previously been shown that two Cp2 molecules are tightly complexed to Kp1 under these conditions and that the predominant species during enzyme turnover is (Cp2 ADPox ) 2 Kp1, but no time dependence of the EPR were reported [15]. The absence of the characteristic EPR signal of Cp2 after 1 h incubation (Fig.…”

“…When turnover is initiated, protons are reduced without a lag, C 2 H 2 is not reduced initially, but becomes an effective substrate after 10 min, [13], and a 30 min lag occurs before N 2 is reduced to NH 3 [14]. There is currently no explanation for this behaviour, but recently we demonstrated that, unlike homologous Kp‐nitrogenase components, Cp2 and Kp1 form a stable 1:1 complex which can be isolated by gel filtration, both in the presence and absence of MgATP [15]. This stable complex is unusual, since although the Cp2 is bound to Kp1, it is reduced as indicated by the presence of a characteristic S =1/2 EPR spectrum of Cp2 in the [4Fe–4S] 1+ oxidation level[16].…”

“…The formation of these novel signals does not arise from the unusual properties of Cp2Kp1‐nitrogenase in forming a stable 1:1 complex [15], since they are also developed by turnover of homologous Kp‐nitrogenase. Fig.…”

Negative cooperativity in the nitrogenase Fe protein electron delivery cycle

“…[1] The prevailing picture of these events is that the two halves of the [MoFe(Fe red (ATP) 2 ) 2 ] complex function independently (1,17,18). However, a previous kinetic study that examined electron delivery by Fe red (ATP) 2 isolated from one organism to MoFe from another organism (a heterologous complex) was inconsistent with this picture and indicated a strong negative cooperativity between reactions at the two halves (19), a phenomenon in which reaction at one-half of an enzyme to some extent suppresses reaction at the other. In the present work, quantitative kinetic measurements of ET, ATP hydrolysis, and P i release in the presteady state, as well as calculations of protein motion, establish whether the two halves of the physiologically relevant homologous [MoFe(Fe red (ATP) 2 ) 2 ] complex between Significance Nitrogenase catalyzes N 2 reduction to ammonia, the largest N input into the biogeochemical nitrogen cycle.…”

“…We considered kinetic schemes in which a randomly selected primary Fe red (ATP) 2 (19), because the overall process is almost instantaneous relative to the other reactions in the scheme.…”