A mechanism accounting for the robust catalase activity in catalase-peroxidases (KatG) presents a new challenge in heme protein enzymology. In Mycobacterium tuberculosis, KatG is the sole catalase and is also responsible for peroxidative activation of isoniazid, an anti-tuberculosis pro-drug. Here, optical stopped-flow spectrophotometry, rapid freeze-quench EPR spectroscopy both at the X-band and at the D-band, and mutagenesis are used to identify catalase reaction intermediates in M. tuberculosis KatG. In the presence of millimolar H 2 O 2 at neutral pH, oxyferrous heme is formed within milliseconds from ferric (resting) KatG, whereas at pH 8.5, low spin ferric heme is formed. Using rapid freeze-quench EPR at X-band under both of these conditions, a narrow doublet radical signal with an 11 G principal hyperfine splitting was detected within the first milliseconds of turnover. The radical and the unique heme intermediates persist in wild-type KatG only during the time course of turnover of excess H 2 O 2 (1000-fold or more). Mutation of Met

Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP ( 13 C, 15 N, and 2 H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both heavy enzymes | transition state coupling | femtosecond dynamics | pre-steady-state chemistry | Born-Oppenheimer enzymes P rotein and atomic motions required for enzymatic catalysis involve a broad range of timescales from the millisecond to the femtosecond. Slower (millisecond) motions are involved in conformational changes that occur during substrate binding and product release (1-4), whereas femtosecond motions (promoting vibrations) are proposed by computational studies to be involved in the chemical step of transition-state formation (covalent bond changes) (5-8). The role of fast (femtosecond) enzyme dynamics in the catalytic cycle has remained elusive and controversial due to the experimental challenge of probing femtosecond motions in enzymes. Previous studies from our laboratory and others have reported that isotopic substitution to create heavy enzymes ( 13 C, 15 N, and 2 H) often slows catalytic site chemistry and, in some cases, alters rate constants for nonchemical steps (9-12). Reduced catalytic site barrier-crossing (the chemical step) in isotopically labeled enzymes supports coupling of local femtosecond motion to transition-state formation and, in some cases, interaction with slower modes (10, 11).In early heavy-enzyme studies, Silva et al. (9) uniformly labeled the amino acid residues of human purine nucleoside phosphorylase (PNP) with 13 C, 15 N, and nonexchangeable 2 H in an attempt to perturb local bond vibrational modes without altering the electrostatics of the enzyme (the Born-Oppenheimer approximation) (13). Heavy PNP demonstrated unchanged steady-state kinetic parameters, kinetic isotope effects, and transition state structure, but the chemical rate was decreased. Transition path sampling with heavy and light PNPs studied reactive trajectories and predicted that femtosecond vibrational motions are less coupled in the heavy enzyme (14).PNP (EC 2.4.2.1) catalyzes the reversible phosphorolysis of 6-oxypurine (and 2′-deoxy)-β-D-ribonucleosides to yield (2-deoxy)-α-D-ribose 1-phosphate and the purine base (15) (Fig. 1). Isotopically labeled PNP expressed from 13 C and 15 N precursors in D 2 O solvent is 9.9% heavier than natural abundance PNP and shows a heavy enzyme kinetic isotope effect (KIE) of 1.36 (k chem light/k chem heavy) (9). Reduced catalytic site ch...