Introduction n 2 1 2 3 4 5 6 n Rhodobacter capsulatus ε −1 −1 7 8 (2+;1+) Pyrococcus furiosus 6 9 10 Materials and methods P. furiosus 11 −1 −1 g 9 10 12 FOR activity was routinely assayed at 80 °C, under anaerobic conditions, with formaldehyde, or glutardialdehyde, as the substrate and 3 mM benzyl viologen as the electron acceptor in 50 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (Epps) buffer, pH 8.4. ε 600 −1 −1 13 ε 400 −1 −1 14 15 d 2 Results Steady-state kinetics 16 1 \documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym} 
\usepackage{amsfonts} 
\usepackage{amssymb} 
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$$
\begin{aligned}
& {\text{E}} + {\text{A}} \;\rightleftharpoons\; {\text{EA}} \;\rightleftharpoons\; \text{E}^{\prime\prime} + {\text{R}} \\
 & \text{E}^{\prime\prime} + {\text{B}} \;\rightleftharpoons\; \text{E}^{\prime\prime} \text{B}\;\rightleftharpoons\; \text{E}^{\prime} + {\text{Q}} \\
 & \text{E}^{\prime} + {\text{C}} \;\rightleftharpoons\; \text{E}^{\prime} \text{C} \;\rightleftharpoons\; {\text{E}} + {\text{P}} \\ 
 \end{aligned} 
$$\end{document} 2 \documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym} 
\usepackage{amsfonts} 
\usepackage{amssymb} 
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$$\begin{aligned} & {\text{E}} + {\text{A}} \;\rightleftharpoons\; {\text{EA}} \\ &{\text{EA}} + {\text{B}} \;\rightleftharpoons\; {\text{EAB}} \;\rightleftharpoons\; {\text{R}} + {\text{EQ}} \\ & {\text{EQ}} \;\rightleftharpoons\; \text{E}^{\prime} + {\text{Q}} \\ & \text{E}^{\prime} + {\text{C}} \;\rightleftharpoons\; \text{E}^{\prime} \text{C} \;\rightleftharpoons\; {\text{E}} + {\text{P}} \\ \end{aligned} $$\end{document} 16 K M K M K M 16 17 3 K siB b 3 \documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym} 
\usepackage{amsfonts} 
\usepackage{amssymb} 
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$$
v = \frac{{V_{{\max }} ab}}
{{K_{{{\text{MA}}}} a + K_{{{\text{MA}}}} b{\left( {1 + \frac{b}
{{K_{{{\text{siB}}}} }}} \right)} + ab}}.
$$\end{document} b v b b v a v a 16 positive 16 a v a b v b 1 2 1 2 Fig. 1 Pyrococcus furiosus  Fig. 2 P. furiosus  K M app V max app K M V max K M app v app 16 4 \documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym} 
\usepackage{amsfonts} 
\usepackage{amssymb} 
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$$
v^{{{\text{app}}}}= \frac{{V_{{\max }} b}}
{{K_{{{\text{MB}}}}+ b}};\quad K^{{{\text{app}}}}_{{{\text{MA}}}}= \frac{{K_{{{\text{MA}}}} b}}
{{K_{{{\text{MB}}}}+ b}};\quad {v^{{{\text{app}}}} } \mathord{\left/
 {\vphantom {{v^{{{\text{app}}}} } {K^{{{\text{app}}}}_{{{\text{MA}}}} }}} \right.
 \kern-\nulldelimiterspace} {K^{{{\text{app}}}}_{{{\text{MA}}}} } = {V_{{\max }} } \mathord{\left/
 {\vphantom {{V_{{\max }} } {K_{{{\text{MA}}}} }}} \right.
 \kern-\nulldelimiterspace} {K_{{{\text{MA}}}} },
$$\end{document} K M V max 1 10 18 K M K M 10 K M V max K M 10 Table 1 Catalytic properties of formaldehyde oxidoreductase determined at 80 °C with formaldehyde as the substrate and ferredoxin or benzyl viologen as the electron acceptor  Electron acceptor Benzyl viologen Ferredoxin K M total formaldehyde 13 ± 2 3.8 ± 0.5 K M  free formaldehyde 71 ± 10 21 ± 3 V max formaldehyde −1 a 4.5 ± 0.5 K M electron acceptor 73 ± 19 14 ± 7 V max electron acceptor −1 32 ± 2 6.2 ± 3.2 K M glutardialdehyde 12 ± 2  V max glutardialdehyde −1 13 ± 1  Materials and methods a 10 V max −1 −1 K M V max K M K M 9 19 K M K M app v app K M K M K M K M 9 K M K M Pre-steady-state kinetics 2+ ε max −1 −1 20 t 3 Fig. 3 a b t c P. furiosus  R. capsulatus 8 4 Fig. 4 P. furiosus  5 t 2 Fig. 5 P. furiosus a b c P. furiosus d P. furiosus  Table 2 Rate constants from pre-steady-state kinetics of formaldehyde oxidoreductase plus formaldehyde or deuterated formaldehyde or formate at 50 °C  UV–vis Fluorescence 2 2 Formate 2 k 1 −1 4.7 1.12 0.51 4.7 k 2 −1 1.9 0.94 −2 1.9 k 3 −1 −2 −2  −2 k 4 −1 −2 −2   21 d 2 d 2 5 2 6 5 5 2 5 t 2 Discussion Steady-state kinetics P. furiosus 6 6 22 15 K M K M The triple-transfer mechanism for three substrates with substrates two and three (ferredoxin) the same is consistent with the experiments. 5 \documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym} 
\usepackage{amsfonts} 
\usepackage{amssymb} 
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$$
\begin{aligned}
& {\text{E}} + {\text{CH}}_{{\text{2}}} {\text{O}} \;\rightleftharpoons\; {\text{ECH}}_{{\text{2}}} {\text{O}} \;\rightleftharpoons\; \text{E}^{\prime\prime} + {\text{CHOOH}} \\
 & \text{E}^{\prime\prime} + {\text{FD}}_{1}\;\rightleftharpoons\; \text{E}^{\prime\prime} \text{{FD}}_{1}\;\rightleftharpoons\; \text{E}^\prime  + {\text{FD}}_{{\text{1}}} ^{\prime }\\
 &  \text{E}^\prime + {\text{FD}}_{2}\;\rightleftharpoons\;  \text{E}^\prime \text{{FD}}_{2}\;\rightleftharpoons\; {\text{E}} + {\text{FD}}_{{\text{2}}} ^{\prime }\\ 
 \end{aligned} 
$$\end{document} K M 10 K M K M K M K M K M Pre-steady-state kinetics Pre-steady-state kinetics studies were performed at 50 °C. This relatively low temperature was dictated by technical limitations of the stopped-flow apparatus. FOR activity at 50 °C is 2.8 times less than at 80 °C, which affords an increased time resolution of enzyme intermediates. Difference spectra from the pre-steady-state data revealed peak shifts and a lack of isosbestic points. These features are an indication that several processes are happening in the first seconds of the reaction. The visible spectrum of the iron–sulfur cubane consists of a single broad line at approximately 400–430 nm; the relatively weakly colored metallopterin is found at different wavelengths. d 2 k 1 −1 k 2 −1 21 2 d 2 x 23 6 Fig. 6 P. furiosus  6 k A −1 k B −1 k C −2 −1 6 k D −2 −1 10 1 k 1 −1 k 2 −1 k 3 −2 −1 10 k obs1 −1 k obs2 −2 −1 6 k 3 k C 6 k cat 7 Fig. 7 P. furiosus A P cube-enclosing circle k A k D k 1 k 4  Under this model the steps observed in the pre-steady-state experiments would actually constitute an activation process. Note that once the steady state of this cycle has been reached, the enzyme reacts sequentially with one formaldehyde and two ferredoxin molecules consistent with the “A+2B” Michaelis–Menten steady-state analysis. 7