The best taxonomically and physiologically characterized examples of H2 pro­ducers are clostridia, but other genera (including bacilli), as well as a microbial flora from anoxic marsh sediments and other environments are known that are capable of the H2 production and either the ABE “solvent” fermentation (chap­ter 6, section 6.3.3), the accumulation of one or more of the ABE trio, carbox­ylic acids (acetic, butyric, etc.) and/or other products (acetoin, 2,3-butanediol, etc.).26

A wide spectrum of carbon sources supports H2 production at rates up to 1,000 ml/hr/g cells at a maximum yield of 4 mol of H2/mol glucose with the stoichiometry:26

C6H12O6 + 2H2O ^ 2CH3COOH + 2CO2 + 4H2

This reaction is sufficiently exothermic (to support microbial growth). The yield of H2 is, however, subject to feedback inhibition by H2, requiring that the partial pres­sure of the gas be kept low to avoid problems with growth rate or a shift to acid production.

Another H2-forming fermentation has butyric acid as its major acidic product: C6H12O6 ^ C3H7COOH + 2CO2 + 2H2

although the molar production of H2 is only half that of acetate-accumulating strains.

The key enzyme in heterotrophic H2 producers is hydrogenase, an enzyme that catalyzes the reoxidation of reduced ferredoxin (Fd), an iron-containing protein reduced by ferredoxin-NAD and pyruvate-ferredoxin oxidoreductases, with the lib­eration of molecular hydrogen (figure 7.5):27

2Fd2+ + 2H+ ^ 2Fd3+ + H2

A summary of hydrogenase-containing bacteria is given in table 7.2.

Hydrogenases are a diverse group of enzymes and are often cataloged on the basis of the metal ion they contain as an essential component of the active site.28 The fastest H2-evolving species under laboratory conditions, Clostridium acetobutylicum, produces two different hydrogenases:29-31

• An iron-containing enzyme, whose gene is located on the chromosome

• A dual-metal (nickel, iron) enzyme whose gene is located on a large plasmid

Glucose

I Butyric Acid

Fructose-1, 6-bisphosphate

1

up to 0.59 ?

up to 0.9

?

up to 1.5

The iron-dependent hydrogenase from C. acetobutylicum has a specific activ­ity eightfold higher than similar enzymes from green algae even when all three enzymes are expressed in and purified from the clostridial host.32 The active sites of iron-dependent hydrogenases may be the simplest such structures yet studied at the molecular level. Of enormous potential importance for the industrial development of hydrogenases is the finding that even simple complexes of iron sulfide and CO mimic hydrogenase action.33 The crucial structure involves two iron atoms with different valency states at different stages of the reaction mechanism (figure 7.6). These findings raise the possibility of rational design of improved hydrogenases by the binding of novel metal complexes with existing protein scaffolds from known enzymes.

In contrast, nickel-iron bimetallic hydrogenases possess complex organometal — lic structures with CO and cyanide (CN-) as additional components, the metal ions bound to the protein via multiple thiol groups of cysteine resides, and an impor­tant coupling between the active site and iron-sulfur clusters.34,35 Multigene arrays are required for the biosynthesis of mature enzyme.36 Nevertheless, progress has been impressive in synthesizing chemical mimics of the organometallic centers that contain elements of the stereochemistry and atomic properties of the active site.37 The enzyme kinetics of nickel-iron hydrogenases remain challenging, and it is pos­sible that more than one type of catalytic activation step is necessary for efficient functioning in vivo.38 39

Such advances in basic understanding will, however, open the door to replacing expensive metal catalysts (e. g., platinum) in hydrogen fuel cells by iron — or iron/ nickel-based biocatalysts — the sensitivity of many hydrogenases to inhibition by O2

OC CO

Fe1 — Fe1———— Fe0 — Fe1

H’H H- ■

I I

Fe11—Fe1 ———— Fe11—Fe1

H+

FIGURE 7.6 Simple organometallic complexes as biochemical mimics of hydrogenase enzymes. (Modified from Darensbourg et al.33)

is a serious drawback but, of the many organism known to produce hydrogenases, some contain forms with no apparent sensitivity to O2 and can function under ambient levels of the gas.40